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PubMed1986

Increased leukocyte diversity and responsiveness to B-cell and T-cell mitogens in cell suspensions prepared by enzymatically dissociating murine lymph nodes.

The isolation of viable follicular dendritic cells (FDCs) from murine lymph nodes requires that the nodes be enzymatically dissociated with collagenase and the protease dispase. The present study was undertaken to compare leukocyte populations derived from enzymatically dissociated and mechanically disrupted lymph nodes. We examined cell viability, cell number, cell types, and the proliferative response to the mitogens LPS, PHA, and Con A. Cells were prepared by taking the inguinal, brachial, axillary, and popliteal lymph nodes from one side of the animals and mechanically disrupting them. The corresponding lymph nodes from the other side of the same animals were digested with enzymes. The enzyme-treated lymph nodes yielded substantially more cells and had a higher percentage of viable cells. Over 40% more viable cells were available for cell culture using the enzyme dissociation method. The numbers of FDCs, macrophages, plasma cells, and fibroblasts were clearly increased. The cells dispersed by enzymatic means responded better than the mechanically dispersed cells to both B-cell and T-cell mitogens. This was especially striking in cultures at lower cell densities. We conclude that the method of cell dissociation has a marked effect on the types of viable cells released and available for culture, as well as on the ability of the cells to respond. We believe the cell types released by enzymatic dissociation and their response in culture more accurately reflect conditions in vivo.

Animals↗
PubMed2001

Variable galactocerebroside expression by human Schwann cells in dissociated and peripheral nerve explant cultures.

It has been well established that rat Schwann cells down regulate their cell-surface expression of galactocerebroside (GalC) in vitro under normal cell culture conditions. To determine whether human Schwann cells exhibit a similar down-regulation of GalC in vitro we examined GalC expression in dissociated human Schwann cell cultures derived from normal adult peripheral nerve. Twenty-four hours post-dissociation up to 63% of human Schwann cells were found to express detectable levels of GalC on their surface whereas less than 8% of the Schwann cells expressed detectable levels of GalC at 14 days post-dissociation. In contrast, after nearly 3 months of peripheral nerve explant culture, greater than 30% of human Schwann cells still retained their GalC expression. A similar pattern was also observed when analyzing Schwann cell purity with dissociated cultures exhibiting a rapid decrease in Schwann cell purity under normal culturing conditions although Schwann cell purity was found to be largely unaffected during the period of peripheral nerve explant culture. In summary, we found there was less variation in both GalC expression and Schwann cell purity with time in peripheral nerve explant cultures than dissociated cultures.

Adult↗
PubMed1989

Neurotransmitter phenotype plasticity in cultured dissociated adult rat dorsal root ganglia: an immunocytochemical study.

Culturing sympathetic ganglion neurons in vitro may modify phenotypic expression of some neurotransmitters. For dorsal root ganglia (DRG), contradictory results have been reported; most studies have used immature material. We have therefore performed a detailed immunocytochemical analysis of the transmitter content of cultured adult rat DRG neurons. To demonstrate possible modifications of neurotransmitter phenotypes, we have compared the results obtained with the same techniques on neurons cultured for 3 days and on freshly dissociated DRG cells. Also, the transmitter profile of cultured neurons was compared with that known from in situ studies. Out of 22 antigens studied, 20 were detected in cultured DRG neurons. All of them were expressed in small and/or intermediate-sized cells. Large neurons only contained CGRP, VIP, NPY, beta-END, ENK, and GABA. The percentage of immunostained neurons varied for the various antisera: less than 10% of cultured neurons were positive for ENK, beta-LPH, beta-END, DYN, VASO, and OXY; 10-30% for SOM, CCK, CAT, and SP; and greater than 30% for NPY, CRF, GLU, NT, VIP, GABA, GRP, CGRP, 5-HT, and TRH. In the latter two groups of transmitters (except CGRP), the proportion of immunoreactive neurons was by far larger in cultured than in freshly dissociated DRG. The most pronounced (greater than 25%) increase in the proportion of positively stained neurons after culturing was observed for the GRP, CRF, TRH, and 5-HT antisera. Serotonin was the only transmitter identified in cultured but not in freshly dissociated cells. These data indicate, on one hand, that various antigens, for example, CAT, GABA, NT, TRH, NPY, beta-LPH, and beta-END, which up to now have not been described in DRG in situ, can be detected immunocytochemically a few hours after dissociation of adult rat DRG. On the other hand, several transmitters, for example, VIP, NPY, SP, GABA, GLU, NT, GRP, CRF, TRH, and 5-HT, are expressed in a significantly higher proportion of cells in cultured than in freshly dissociated preparations. This might reflect a change in the phenotypic expression of transmitters due to the new environment generated by the culture conditions, a hypothesis that can be tested by measuring specific mRNA levels. Moreover, considering the plasticity and multipotentiality of their transmitter phenotype, cultured adult DRG neurons might represent an interesting material for autografts into the injured central nervous system.

Animals↗
PubMed1992

In search of the central respiratory neurons: I. Dissociated cell cultures of respiratory areas from the upper medulla.

Dissociated cells from the areas of the nucleus ambiguus and the nucleus tractus solitarius obtained by tissue punch or block dissection from coronal slices of the medulla at the level of the obex were cultured from fetal rats at 18 to 21 days gestation. The dissociated neurons were plated either directly in vitrogen-coated 35 mm tissue culture dishes or in such dishes which had been seeded with subcultures of cortex- or medulla-derived astrocytes. After the astrocytes reached confluency and were treated with an antimitotic agent, dissociated nucleus ambiguus or nucleus tractus solitarius was plated at 0.5-1.0 x 10(6) cells per dish. Neurons grew well on monolayers of medullary or cortical astrocytes, but survived poorly on vitrogen-coated dishes without a cellular substrate. Rat medulla was preferred as the source of astrocytes. Tissue dissociation with papain rather than trypsin produced less cellular debris, and the neuronal yield from the tissue was higher. The neuronal population was heterogenous in morphology including small and large bipolar, pyramidal, and multipolar cells. Neurons sensitive to CO2 and/or low pH (Rigatto et al., J Neurosci Res 33:590-597, 1992) did not appear to have any definitive morphologic characteristics, but most were multipolar. These neurons stained well with antibodies to neuron-specific enolase and Fragment C of tetanus toxin, but not to choline acetyltransferase (ChAT). These findings suggest that neurons possibly responsible for the central regulation of respiration can be maintained for several weeks in dissociated cell culture, providing a system for neurotransmitter, electrophysiological, and morphological studies.

Animals↗
PubMedDate not supplied

The role of electron capture dissociation in biomolecular analysis.

The introduction of electron capture dissociation (ECD) to electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) constitutes a significant advance in the structural analysis of biomolecules. The fundamental features and benefits of ECD are discussed in this review. ECD is currently unique to FT-ICR MS and the fundamentals of that technique are outlined. The advantages and complementarity of ECD in relation to other tandem mass spectrometry (MS/MS) techniques, such as infrared multiphoton dissociation (IRMPD) and sustained off-resonance collision-induced dissociation (SORI-CID), are discussed. The instrumental considerations associated with implementation of ECD, including activated ion techniques and coupling to on-line separation techniques, are covered, as are the allied processes electronic excitation dissociation (EED), electron detachment dissociation (EDD), and hot electron capture (HECD). A major theme of this review is the role of ECD in proteomics, particularly for characterization of post-translational modifications (phosphorylation, glycosylation, carboxyglutamic acid, sulfation, acylation, and methionine oxidation) and the top-down approach to protein identification. The application of ECD to the analysis of polymers, peptide nucleic acids, and oligonucleotides is also discussed.

Cyclotrons↗
PubMed1996

Evidence for the dissociation of the hepatobiliary MRI contrast agent Mn-DPDP.

These experiments assessed and quantitated the release of free manganese Mn++ from the hepatobiliary contrast agent Mn-DPDP (manganese dipyridoxal diphosphate), using several magnetic resonance techniques (EPR spectroscopy, 31P-NMR spectroscopy, and relaxometry) to differentiate between free Mn++ and Mn++ in complexes in various preparations. The presence of calcium and magnesium in physiological concentrations in aqueous solutions induced the release of Mn++ from the complex, as did incubation of the complex in liver homogenates. After intravenous injection of 15 mumol/kg of Mn-DPDP, both EPR and 31P-NMR spectroscopy demonstrated that Mn-DPDP is partly dissociated (approximately 25%) in the liver. By comparing in vitro and ex vivo data from the liver, we concluded that the dissociation of Mn-DPDP occurs primarily in the liver, whereas a minor portion of the dissociated. Mn found in the liver comes from dissociation of the complex in the blood. Most of the dissociated Mn in liver becomes bound to macromolecules and is responsible for the enhancement of relaxivity observed with this agent.

Animals↗
PubMed2003

Cone voltage induced in-source dissociation of glucuronides in electrospray and implications in biological analyses.

Glucuronidation is a common mechanism in drug metabolism. In-source dissociation of glucuronides in electrospray generates fragment ions identical to those of the precursor ions of the original drugs. The effect of experimental parameters on the process was investigated in the present study using both commercially available compounds and glucuronides generated from microsomal glucuronidation incubations. It was found that cone voltage was the most critical parameter contributing to in-source fragmentation of both O- and N-glucuronides, whereas both the desolvation temperature and the source temperature had little effect. Additionally, the extent of in-source dissociation varied for different glucuronides and could be minimized by lowering cone voltage. As demonstrated in real examples, minimizing in-source dissociation can lead to higher sensitivity in detecting glucuronides in biological samples. In addition, product ions resulting from in-source dissociation of glucuronides potentially interfere with accurate determinations of corresponding drug levels if chromatographic separation is not adequate. For cases in which chromatographic separation of glucuronides from the original drugs is not readily achieved or high-throughput analyses are desired, interference caused by in-source dissociation can usually be eliminated simply by using lower cone voltage. This approach has been proven to be effective in the analysis of more than 100 glucuronides generated from in vitro microsomal incubations.

Electrochemistry↗
PubMed2005

Dissociation of gas-phase dimeric complexes of lactic acid and transition-metal ions formed under electrospray ionization conditions; the role of reduction of the metal ion.

Dimeric complex ions of the type [M(A-H)A]+, where M=metal ion (Co, Ni, Cu, and Zn) and A=ligand (lactic acid, methyl lactate or ethyl lactate), were generated in the gas phase under electrospray ionization conditions. The collision-induced dissociation spectra of [M(A-H)A]+ ions were recorded to study the behaviour of ligand and metal ions in decomposition of these dimeric complex ions. Based on the fragmentation pathways observed for complex ions of lactic acid, it is found that both the carboxylic and hydroxyl groups of lactic acid are involved in the complex formation following displacement of a proton by the metal ion. The dimeric complex ions of Co, Ni, and Zn dissociated to yield similar types of ions, whereas that of Cu behaved differently. The dissociations of Co-, Ni-, and Zn-bound dimeric complexes involved losses of neutral molecules while keeping the oxidation state of the metal ion unchanged. However, elimination of radicals is found in the dissociation of dimeric complex ions of Cu, and the oxidation state of copper is reduced from Cu(II) to Cu(I) in the resulting fragment ions. The deprotonated ligand is involved in the fragmentation pathway of Cu complexes, whereas it is intact in other complexes. The oxidation state of the metal ion, nature of the ligand, and site of attachment to the metal ion are found to control the dissociation of these dimeric complex ions.

Dimerization↗
PubMed1998

Calcium-induced quenching of intrinsic fluorescence in brain myosin V is linked to dissociation of calmodulin light chains.

Myosin V isolated from chick brain (BM V) is a multimeric protein of about 640 kDa consisting of two intertwined heavy chains of 212 kDa and multiple light chains of 10 to 20 kDa. A distinctive feature of the heavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin light chains. The actin-activated MgATPase has been shown to require >/=1 microM Ca2+ for full activity, and evidence points to a myosin-linked regulatory system where the CaM light chains participate as modulators for the Ca2+ signal. Still, the precise mechanism of Ca2+ regulation remains unknown. In the present study we have used the intrinsic tryptophan fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation from the BM V molecule. The fluorescence intensity decreases approximately 17% upon addition of sub-micromolar concentrations of Ca2+ (K0.5 = 0.038 microM). This decrease in fluorescence, which is dominated by a conformational change in the heavy chain, can be reversed by addition of 1, 2-di(2-aminoethoxy)ethane-N,N,N',N'tetraacetic acid (EGTA) followed by an excess of CaM, but not by addition of EGTA alone. Gel filtration of native BM V using HPLC shows that CaM is partially dissociated from the heavy chain in EGTA and dissociates further upon addition of sub-micromolar concentrations of Ca2+. These observations suggest that the affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with Ca2+ and that the Ca2+ concentration required for this effect is lower than that needed to activate acto-BM V. Using a cosedimentation assay in the presence of actin, we also observe partial dissociation of CaM when Ca2+ is absent, but now the addition of Ca2+ has a biphasic effect: sub-micromolar Ca2+ concentrations lead to reassociation of CaM with the heavy chain, followed by dissociation when Ca2+ exceeds 5-10 microM. Thus, the binding of CaM to BM V is affected by both actin and Ca2+.

Actins↗
PubMed1994

An electrophoretic method to detect cold-induced dissociation of proteins in crude extracts of higher plants.

The phenomenon of cold denaturation has been firmly established recently in several proteins. Some multimeric enzymes from plant origins are believed to dissociate under some circumstances in the cold. To determine the presence and number of soluble, non-membrane-bound protein that undergoes cold-induced dissociation in plants, we have devised a special two-dimensional polyacrylamide gel electrophoretic method using two native gradient gels. Examination of the gel run at 0 degrees C in the second dimension showed the presence of four cold-dissociated proteins running below the diagonal and staining intensely with silver, in the extract of maize leaves. The electrophoretic method described here is expected to be a convenient way to detect cold-induced dissociation of soluble proteins in crude extracts of various tissues. It is also possible to estimate roughly the molecular weights of both the cold-dissociated subunit and the native protein from which it is derived.

Cold Temperature↗
PubMed1993

Dissociation of the peptide/MHC class I complex: pH dependence and effect of endogenous peptides on the activation energy.

Dansylated peptides were used to characterize the dissociation of peptides from a recombinant class I major histocompatibility complex protein. Dissociation of endogenous, low-affinity peptides from the class I molecule Kd had an activation energy of 6.78 +/- 0.64 kcal/mol in the 14 to 26 degrees C temperature range, but there was a break in the Arrhenius plot between 12 and 14 degrees C. Dissociation of a dansylated, high-affinity peptide had an activation energy of 20.24 +/- 1.69 kcal/mol, and there was similarly a break in the plot. Both direct interactions between peptide and the class I heavy chain and indirect effects of the peptide affinity on the extent of light chain association with heavy chain may contribute to the difference in activation energies, while the break in the Arrhenius plots implies a temperature-dependent conformational change. Dissociation was also slowest at neutral pH, but the peptide/class I complex dissociated rapidly at pH greater than 9 and less than 5, suggesting that endocytosed class I proteins would most likely lose their bound peptides at the acidic pH of endosomes.

Amino Acid Sequence↗
PubMed1997

Dissociation of nitric oxide from soluble guanylate cyclase.

Kinetic studies of soluble guanylate cyclase complexed with nitric oxide prove that NO dissociation in the presence of the substrate GTP and Mg2+ is as much as 50 times faster than in their absence. In the presence of those two reagents the dissociation rate constant is k(obs) = 0.04 +/- 0.01 s-1 at 20 degrees C, which is by far the fastest NO dissociation rate constant ever reported for a ferrous heme protein. Extrapolated to 37 degrees C, this corresponds to a half life of about 5 s for NO dissociation from soluble guanylate cyclase at physiological conditions, which is presumably fast enough to account for deactivation of the enzyme in biological systems. Dissociation rate constants are also reported for a variety of other reagent conditions.

Animals↗
PubMed1999

Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.

Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids. The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.

Allosteric Regulation↗
PubMed1985

Consequences of the O2-CO2 dissociation curves for gas exchange in avian and mammalian lung at various altitudes.

Gas exchange in avian and mammalian lung was studied with computer simulations using mutually dependent O2-CO2 dissociation curves. The mammalian lung was simulated with an ideal mixing unit. The model of the avian lung consisted of 25 ideal mixing units placed in series with respect to the ventilation but placed in parallel with respect to the perfusion (cross-current arrangement). The consequence of the right shift in the avian oxygen dissociation curve, if compared with the human dissociation curve, was studied by application of both dissociation curves to the avian lung model. The influence of a decreased tension of oxygen in the inspired air was studied under conditions of constant PvCO2 (48 mmHg), PvO2 (30 mmHg), RQ (0.70) and approximately constant levels of PaCO2. At sea-level the avian lung needs 80 per cent of the alveolar ventilation and 64 per cent of the cardiac output to transport the same amount of gas if compared with the mammalian lung. The difference in the need for cardiac output resulted from the right shift in the dissociation curve of avian blood. Changing PIO2 from 115 mmHg (2000 m) to 100 mmHg (3000 m) showed a dramatic difference in the performance of the two lung models. The mammalian lung model needed a more than eight fold increase in cardiac output whereas the avian lung model needed less than a three fold increase to maintain a RQ of 0.7. This difference could be ascribed to the Haldane effect in combination with the cross-current arrangement. The Haldane effect was also responsible for an end expired CO2 tension which exceeded the mixed venous CO2 tension.

Adaptation, Physiological↗
PubMed1983

Tests of the ribosome editor hypothesis. II. Relaxed (relA) and stringent (relA+) E. coli differ in rates of dissociation of peptidyl-tRNA from ribosomes.

Derivatives of isogenic stringent (relA+) and relaxed (relA) strains of Escherichia coli were compared in respect of rates of the dissociation of peptidyl-tRNA from ribosomes during protein synthesis. The derivatives both contained a mutant pth gene which rendered temperature-sensitive their peptidyl-tRNA hydrolase (E.C. 3.1.1.29) activities. After shifting from permissive 30 degrees C to non-permissive 40 degrees C, dissociated peptidyl-tRNA accumulated and was assayed chemically or by its cytotoxic effects. In unperturbed (except for the temperature shift) cultures the relA strain accumulated peptidyl-tRNA significantly more slowly than did its relA+ isogenic cousin. Both strains responded approximately equally to non-lethal doses of erythromycin or to starvation for amino acids. Both these perturbations enhanced the dissociation and accumulation of peptidyl-tRNA. While growing at 30 degrees C, both strains responded significantly to a nutritional downshift from growth in medium containing glucose plus amino acids to growth in medium containing only amino acids. Taken together the results suggested that different intracellular concentrations of ppGpp in unperturbed cells, attributable to the different relA alleles, could account for the differences in dissociation and accumulation of peptidyl-tRNA. Our observation of a lower rate of dissociation of peptidyl-tRNA in the relA strain, coupled with the reported lower intracellular ppGpp and lower accuracy of protein synthesis, is consistent with the idea that relA strains have less efficient ribosomal editing of erroneous peptidyl-tRNA.

Amino Acids↗
PubMed1978

CO2 dissociation curves of oxygenated whole blood obtained at rest and in exercise.

In vitro CO2 dissociation curves for oxygenated whole blood were determined in 19 healthy male subjects at rest and during submaximal and maximal bicycle work. Hemoglobin concentration and blood lactate increased with increasing work load and accordingly buffer value of the whole blood increased while bicarbonate and Base Excess (BE) decreased, resulting in a downward shift of the CO2 dissociation curve during exercise. Despite the marked increase in buffer values of the blood, the slopes of the CO2 dissociation curves during exercise were found to be about the same as those obtained at rest. It was inferred that the increasing effect of increased buffer value, on the dissociation slope, was essentially compensated by the decreasing effect of diminished bicarbonate content. The advantages of this relatively constant CO2 dissociation slope for the indirect measurement of cardiac output by the Fick principle are discussed.

Adult↗
PubMedDate not supplied

Benign nature of atrial dissociation in pediatric patients.

Atrial dissociation is defined electrocardiographically by the presence of an ectopic atrial rhythm depolarizing independently of the basic sinus rhythm, and is characterized by entrance and exit block. Atrial dissociation is most often observed as an ominous sign in adults with severe myocardial dysfunction. We have observed atrial dissociation in 3 pediatric patients, aged 5 to 22 years, who were essentially asymptomatic from this arrhythmia. All 3 patients are well, without therapy for atrial dissociation, during a follow-up period of 1 to 5 years. The benign nature of atrial dissociation in the pediatric patient as compared with the adult is stressed.

Adolescent↗
PubMed1989

Automated selective dissociation of cells from different regions of multicellular spheroids.

In this report we describe a new apparatus which has been developed for the automated selective dissociation of multicellular spheroids into fractions of viable cells from different locations in the spheroid. This device is based on the exposure of spheroids to a 0.25% solution of trypsin under carefully controlled conditions, such that the cells are released from the outer spheroid surface in successive layers. Study of the spheroid size, number of cells per spheroid, and sections through the spheroid with increasing exposure to trypsin demonstrate the effectiveness of this technique. The technique has been successfully used on spheroids from five different cell lines over a wide range of spheroid diameters. We also present data detailing the effect of varying the dissociation temperature, the mixing speed, the trypsin concentration, and the number of spheroids being dissociated. The new apparatus has several advantages over previous selective dissociation methods and other techniques for isolating cells from different regions in spheroids, including: a) precise control over dissociation conditions, improving reproducibility; b) short time to recover cell fractions; c) ability to isolate large numbers of cells from many different spheroid locations; d) use of common, inexpensive laboratory equipment; and e) easy adaptability to new cell lines or various spheroid sizes. Applications of this method are demonstrated, including the measurement of nutrient consumption rates, regrowth kinetics, and radiation survivals of cells from different spheroid regions.

Cell Adhesion↗