Cryptofluorescent analogs of cobalamin coenzymes: synthesis and characterization.

Upper-axial (beta-position) ligand analogs of the B12 coenzymes 5'-deoxy-5'-adenosylcob(III)alamin and methylcob(III)alamin have been synthesized by reaction of the 5'-chloro-5'-deoxy derivatives of fluorescent nucleosides (1,N6-ethenoadenosine, formycin, 2-amino-nebularine, and 2,6-diaminonebularine) and a fluorescent alkyl halide (dansylamidopropyl chloride) with cob(I)alamin. These analogs were nonfluorescent, but fluorescent products could be generated by photolysis or cyanolysis of the carbon-cobalt bonds. Under anaerobic and aerobic conditions, the major fluorescent photolysis products of 1,N6-ethenoadenosylcob(III)alamin were 1,N6-etheno-5',8-cyclic-5'-deoxyadenosine, and the 5'-aldehyde of 1,N6-ethenoadenosine, respectively. The cryptofluorescent property of these analogs was utilized to follow the kinetics of aerobic photolysis. First-order rate constants determined by this method were comparable to those obtained spectrophotometrically [via appearance of of aquacob(III)alamin]. Pseudo-first-order rate constants determined fluorometrically for the cyanolysis (at 25 degrees C) of 1,N6-ethenoadenosylcob(III)alamin, 2,6-diaminonebularinylcob(III)alamin, 2-aminonebularinylcob(III)alamin and formycinylcob(III)alamin were 5.8 X 10(-2), 2 X 10(-2), 1.8 X 10(-2), and 3 X 10(-5) min-1, respectively; values in good agreement were obtained spectrophotometrically (via appearance of dicyanocobalamin). Dansylamidopropylcob(III)alamin was stable in the presence of cyanide. The nucleoside alpha-ribazole is fluorescent in the free state but nonfluorescent when present as the lower axial (alpha-position) ligand in cobalamin coenzymes. Thus fluorescence of ligands in both the alpha- and beta-positions of cobalamins is quenched, probably as a result of intramolecular energy transfer between the ligands and the nonfluorescent corrinoid.     

Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation.

The conversion of cyanocobalamin to adenosyl- and methylcobalamin is impaired in cobalamin-deficient cultured human glial cells. In contrast cultured human skin fibroblasts retained their ability to synthesize coenzyme forms when grown in cobalamin-deficient medium. Cells were pre-conditioned by growing in cobalamin-deficient media for six weeks and then subcultured in medium containing either free or transcobalamin II-bound 57Co-cyanocobalamin. Although both coenzyme levels were low in cobalamin-deficient glial cells, the decrease in methylcobalamin was more marked than that of adenosylcobalamin. Methionine synthase and Cb1 reductase activities were markedly decreased in cobalamin-deficient glial cells but were unchanged in fibroblasts cultured in cobalamin-deficient medium. Our data suggest that in glial cells, cobalamin coenzyme synthesis and function is exquisitely sensitive to short-term cobalamin deprivation. Glial cells apparently synthesize and secrete transcobalamin II since antibodies directed against the transport protein inhibit the uptake of free cobalamin.     

Expression of fetal thymidine kinase in human cobalamin or folate deficient lymphocytes.

In extracts of peripheral blood lymphocytes of cobalamin or folate deficient patients thymidine kinase activity is increased three fold and exhibits properties of the fetal isoenzyme. Appropriate vitamin therapy results in reduction of this activity to normal levels and change from fetal to adult isoenzyme. The occurrence in cobalamin or folate deficiency of fetal thymidine kinase activity in non proliferating human lymphocytes is unique and may reflect events in the deficient marrow lymphoid progenitor cells.     

Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor

5-Methylcytosine synthesis in DNA involves the transfer of methyl groups from S-adenosyl-methionine to the 5'-position of cytosine through the action of DNA (cytosine-5)-methyltransferase. The rate of this reaction has been found to be enhanced by cobalt ions. We therefore analyzed the influence of vitamin B12 and related compounds containing cobalt on DNA methylation. Vitamin B12, methylcobalamin, and coenzyme B12 were found to enhance significantly the de novo DNA methylation in the presence of S-adenosylmethionine for concentrations up to 1 microM, but at higher concentrations these compounds were found to inhibit DNA methylation. Methylcobalamin behaves as a competitive inhibitor of the enzymatic methylation reaction (Ki = 15 microM), the Km for S-adenosylmethionine being 8 microM. In addition, the use of radioactive methylcobalamin shows that it can be used as a methyl donor in the de novo and maintenance DNA methylation reactions. Thus, two DNA methylation pathways could exist: one involving methylation from S-adenosylmethionine and a second one involving methylation from methylcobalamin.     

Rapid analysis of cobalamin coenzymes and related corrinoid analogs by high-performance liquid chromatography

Cobalamin coenzymes and a series of related corrinoid analogs have been analyzed by high-performance liquid chromatography on reverse-phase C₈ and C₁₈ columns using both isocratic and gradient elution systems and 254-nm absorbance detection. In the isocratic mode, retention times for sulfitocobalamin, cyanocobalamin, methylcobalamin, adenosylcobalamin, and aquacobalamin on a LiChrosorb C₈ column were 1.1, 1.6, 2.2, 2.9, and 4.7 min, respectively. In the gradient mode, corresponding retention times were 9.7, 10.2, 12.8, 11.4, and 9.0 min. Closely related structural analogs of adenosylcobalamin such as 1,N⁶-ethenoadenosylcobalamin, formycinylcobalamin, and 2,6-diaminonebularinylcobalamin were clearly resolved from naturally occurring cobalamins by isocratic elution. In the gradient system, the order of elution of cobalamins was related to the hydrophobicity of the upper-axial ligand. This was demonstrated by determining the retention times of aminoalkylcobalamin homologs (C₂, 9.8; C₃, 10.5; C₅, 11.2; C₈, 12.8; and C₁₁, 14.9 min). The usefulness of this method was demonstrated by analyzing ⁵⁷Co-labeled cobalamins present in extracts of Lactobacillus leichmannii and murine leukemia L1210 cells.     

In vitro response of subpopulations of human lymphocytes. II. DNA synthesis induced by anti-immunoglobulin antibodies.

A significant and constant increase in DNA synthesis was observed in human lymphocytes cultured in the presence of purified anti-immunoglobulin antibodies specific for human IgG, IgA, and IgM. This has been found in cultures of lymphocytes isolated from blood, tonsils, spleen, and lymph nodes. The optimal culture conditions for blood and tonsil lymphocytes were determined. As a rule 6-day cultures containing 2 x 10(6) cells/ml and 100 mug/ml of antibody yielded the highest 3H-thymidine uptake. Purified T cell cultures could not be stimulated, whereas a low response could be observed in most of the purified B cell cultures. Optimal culture conditions were the same for the B and total tonsil lymphocytes. However, when the purified B cells were totally depleted of T cells, no response was observed. A T and B cell synergy has been demonstrated by supplementing B cell cultures with purified T cells, whether treated or not with mitomycin. These experiments indicated a permissive and potentiating effect of T cells on the B cell response. Cultures containing mitomycin-treated B cells and purified T cells (mB + T) could be stimulated by a-Ig, thus indicating a T cell proliferation. In keeping with this finding was the observation of an increased response of total lymphocytes supplemented with T cells but not with B cells. Adherent cells are necessary for an optimal response to a-Ig; they enhanced the B cell proliferation observed in (Tm + B) cultures and suppressed the response of T cells in (T + Bm) cultures.     

Analysis of the nucleoside moiety of cobalamin and cobalamin analogues using gas chromatography-mass spectrometry.

Existing techniques for identification of cobalamin and cobalamin analogues generally use the intact molecule during characterization with somewhat ambiguous results. In this study a method is described for the identification of the nucleoside in the lower axial ligand of cobalamin and a variety of naturally occurring cobalamin analogues that differ from cobalamin in the base that is present in the nucleoside. Cobalamin and cobalamin analogues were isolated from biological samples by affinity chromatography using R-protein-Sepharose columns. The nucleosides of the lower axial ligand were then hydrolyzed and isolated by column chromatography using a mixed bed column. Nucleosides were oxidized with periodate and reduced with borohydride. After reisolation, the t-butyldimethylsilyl derivatives were prepared and analyzed using gas chromatography/mass spectrometry with selected ion monitoring. A stable isotope internal standard of cobalamin was biosynthetically produced and used to quantitate cobalamin in rabbit kidney. Cobalamin analogues were also shown to be present in rabbit kidney, but they contain the 5,6-dimethylbenzimidazole nucleoside (alpha-ribazole) in the lower axial ligand, indicating that these analogues differ from cobalamin in the corrin ring region of the molecule.     

Control of phosphoribosylpyrophosphate synthesis in human lymphocytes.

In an attempt to determine if arginyl residues play a role in sulfate transfer reactions, we studied the effects of 2,3-butanedione and phenylglyoxal, both chemical modifying agents for arginyl residues, on phenol-sulfotransferase. Both reagents produced rapid inactivation of the enzyme, with the inactivation following pseudo-first order kinetics. The rate of inactivation was dependent upon the concentration of the chemical modifier. Competition studies showed that inclusion of 3′-phosphoadenosine-5′-phosphosulfate during the preincubation step protected the enzyme from inactivation. The results suggest a possible role for arginyl residues as anionic recognition sites for sulfate transfer reactions.     

Unscheduled synthesis induced by thiophosphamide in human lymphocytes

An unscheduled DNA synthesis in human nonreplicative lymphocytes is shown to follow cell exposure to thiophosphamide at a dose level of 1-10 microgram/ml. Incorporation of 3H-thymidine in the absence of the mutagen is probably due to spontaneous reparative processes. Significant variations are found of spontaneous and thio-phosphamide-induced levels of a reparative DNA synthesis in normal individuals.     

In vitro synthesis of human IgE by neonatal lymphocytes: enhancing effect of interferon-gamma

The present study demonstrates that neonatal human lymphocytes that are incapable of producing IgG and IgA antibodies may differentiate into IgE-secreting cells under the influence of IL4. Indeed, the addition of recombinant IL4 to cultures of unfractionated umbilical cord blood mononuclear cells (CBMC) induces a dose-dependent synthesis of IgE but not of the other classes of Ig. Moreover, IgE-secreting B lymphoblastoid cell lines can be derived from neonatal lymphocytes costimulated with EBV and IL4. Comparison of the mechanisms regulating the in vitro IgE synthesis by adult and neonatal lymphocytes indicates that in most cases IFN-gamma markedly potentiates the IgE synthesis in CBMC cultures whereas it has a reverse effect on adult lymphocytes. These reciprocal effects of IFN-gamma are specifically blocked by a neutralizing mAb to IFN-gamma; they are dose-dependent and they are observed when IFN-gamma is added at the initiation of the culture or shortly thereafter. Moreover, in a small number of cases IFN-gamma may also potentiate IgE synthesis by adult lymphocytes. The potentiation or the suppression of IgE synthesis by IFN-gamma is not explained by a differential effect of IFN-gamma on the production of soluble CD23 (sCD23); indeed in both cases IFN-gamma slightly increases the IL4-induced production of sCD23. Moreover, the spontaneous and the IL4-induced production of sCD23 by CBMC is comparable to that of adult lymphocytes. The IgE response is dependent upon the expression of Fc epsilon RII (CD23) inasmuch as it is specifically blocked by anti-CD23 mAb.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.