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Biomedical subjects

J A Begley

Publications and source records attributed to J A Begley.

At least 19 recordsLinked to original sources
PubMed1989

The nature of the defect in cobalamin G mutation.

Cobalamin G mutation (cblG) typically presents as a severe megaloblastic anemia during the first few weeks of life. The anemia responds completely to treatment with high doses of Cbl but the neurologic manifestations respond more slowly and not always completely. Cultured fibroblasts from two affected infants and virus-transformed lymphoblasts from one of the infants expressed the following: poor growth in the absence of methionine, the ability to take up and internalize Cbl bound to transcobalamin II, impaired synthesis of methionine from homocysteine, the ability to bind incoming Cbl to the respective coenzymes, but an inability to synthesize methylcobalamin in spite of a normal capacity to synthesize adenosylcobalamin. The in vitro activity of the methyltransferase dependent on methylcobalamin of cell extracts varied from low to high depending on the conditions of culture and assay. The cblG cells were unusually sensitive to the concentration of adenosylmethionine in the assay. More adenosylmethionine was required by cblG cells to achieve the same level of enzyme activity as control cells and high concentrations of adenosylmethionine did not suppress activity as in control cells. It was postulated that the defect in cblG is in the metabolism of adenosylmethionine, an essential substance for the synthesis of methionine from homocysteine.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1988

The role of transcobalamin II in the methionine dependency of human lymphocytes.

Neither normal human B lymphoblasts (RPMI 6410) transformed by the EB virus nor human peripheral blood lymphocytes (PBL) stimulated by a mitogen replicated well when the methionine (Met) of the medium was replaced with homocysteine (Hcy). Cbl bound to human transcobalamin II (TC II) substantially increased cell division over that observed when the Cbl of the medium was in the free form. Although, as expected, the TC II enhanced the cell entry of Cbl 1000-fold, this was not the basis of the TC II effect. Through adjustment of the respective concentrations of free Cbl and TC II-Cbl in the medium, equal amounts of Cbl entered the cell, yet the TC II effect persisted. TC II-Cbl did not restore cell division in the absence of Met by virus-transformed lymphoblasts from a child with defective Met synthesis from Hcy. The TC II did not act by enhanced induction of the Cbl-dependent methionine synthase activity of cell extracts but the ability of intact cells to produce Met from Hcy by the Cbl-dependent process appeared to have a role in the TC II effect.

Cells, Cultured↗
PubMed1987

Cyclic activity of the receptors of cobalamin bound to transcobalamin II.

The activity of receptors specific for human transcobalamin II-Cobalamin (TC II-Cbl) were measured in virus-transformed lymphoblasts, hepatocytes (hepatoma) and diploid fibro lasts. In all three types of human cells the receptor activity increased as cells went from a resting phase to the most actively dividing phase. Receptor activity declined as cell division slowed. The changes in activity of lymphoblasts and hepatocytes were produced by changes in receptor number and not by changes in affinity between receptors and TC II-Cbl. The basis of the change in fibroblasts was not clear. The Cbl-dependent methionine synthetase activity of fibroblasts, in contrast, tended to be greatest when the cultures were confluent and replication had slowed. As the fibroblasts became senescent the receptor activity for TC II-Cbl declined and the fluctuations with the phase of the cell were blunted. However, the release of apo TC II from the cells was maintained. These observations must be taken into consideration when the respective cells are used as models. Even more important are the implications of the observations of the changes in receptor activity for TC II-Cbl for the regulation of the entry of Cbl into cells.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1986

A patient with the inability to maintain in vivo levels of bound cobalamin (Cbl) and manifestations of tissue deficiency of Cbl.

A 39-year-old woman presented with mild anemia, glossitis, an increased MCV, a low serum cobalamin (Cbl) (vitamin B12), mild tissue deficiency of Cbl, but with neither malabsorption of Cbl, impaired intake, nor deficiency of or inactivity of transcobalamin II (TC II). Because of a persistently low holo-TC II (TC II carrying Cbl as the circulating complex of TC II-Cbl), much of the evaluation was focused on the patient's TC II. Her TC II promoted the uptake of Cbl, reacted with anti-TC II, and bound Cbl in vitro. A test dose of 200 micrograms of cyanocobalamin (CN-Cbl) i.m. increased her holo TC II to levels higher than those in healthy persons, but with a much more abrupt fall to a subnormal level. Two milligrams of CN-Cbl i.m. followed by 100 micrograms i.m. monthly failed to maintain normal amounts of circulating TC II-Cbl or to overcome the tissue deficiency of Cbl. One milligram i.m. weekly or daily p.o. corrected both. The low holo TC II was considered to be responsible for the clinical expression and may have been primary to the reduced amounts of total and holo R binder of Cbl in the circulation. This study of a newly recognized defect points out the need for circulating holo TC II, a rational use of pharmacologic amounts of Cbl, and a possible interrelationship between TC II and the R binder of Cbl.

Adult↗
PubMed1986

Methionine dependency of cultured human lymphocytes.

Human peripheral blood lymphocytes stimulated with phytohemagglutinin and a lymphocyte model consisting of the RPMI 6410 cell, a human virus-transformed B cell, required added methionine (Met) for growth of the cultures. This failure to meet all needs for Met via endogenous synthesis, which is characteristic of oncogenic transformation, occurred even in the presence of adequate homocysteine, methylfolate (5-CH3-H4PteGlu) and cobalamin (Cbl)-dependent methionine synthetase activity. Folinic acid (5-CHO-H4PteGlu), which provides available folate independently of Cbl, improved growth only slightly in the absence of Met. Free Cbl at 222 nM, an amount great enough to alter other intracellular events, failed to increase growth in the absence of Met, but 0.22 nM Cbl bound to transcobalamin II did, however, enhance growth.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1986

Methionine synthetase activity of human lymphocytes both replete in and depleted of vitamin B12.

The activity of the enzyme methionine synthetase (MS) (methyltetrahydrofolate:homocysteine methyltransferase) (EC 2.1.1.13) was measured in human lymphocytes of various types and cobalamin (vitamin B12) status. Total and holo MS activity was low in unstimulated peripheral blood lymphocytes from persons with tissue deficiency of cobalamin, but not in cells from those with low serum cobalamin levels for other reasons. The MS activity of the lymphocyte was increased by treatment of the patients with vitamin B12. The number of lymphocytes was often low or low normal in the circulation of those deficient in cobalamin. Holo MS activity was low in an established line of human B cells, RPMI 6410 cells, depleted of cobalamin. The total and holo MS activity of both RPMI 6410 cells, replete or depleted, and lymphocytes stimulated in culture was increased by cobalamin in vitro; 222 nmol/L free cobalamin was roughly the equivalent of 0.22 nmol/L cobalamin bound to transcobalamin II. Both lymphocytes and RPMI 6410 cells required folate for growth and could meet these needs via methylfolate, homocysteine, and the cobalamin-dependent MS reaction. Depleted RPMI 6410 cells, however, used cobalamin in some way in addition to the provision of available folate from methylfolate. The consequences of the reduced MS activity in deficient cells could include a reduction in available folate with diminished capacity for clonal expansion of lymphocytes in reaction to infection and impairment of essential methylations including those of protein synthesis. The prompt induction of MS activity by cobalamin, especially in the in vitro model, suggests an effect of therapeutic vitamin B12 well in advance of the numerical increase in cells of the blood.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1985

Synthesis of transcobalamin II by cultured human hepatocytes.

Cultured HepG2 cells, derived from a human hepatoma synthesized and released unsaturated, immunoreactive transcobalamin II. Synthesis was confirmed by the blocking with inhibitors of protein synthesis and by incorporation of tritiated leucine into transcobalamin II.

Apoproteins↗
PubMed1985

The metabolism of cobalamin bound to transcobalamin II and to glycoproteins that bind Cbl in HepG2 cells (human hepatoma).

The binding, internalization, processing and release of labeled cyanocobalamin (CN[57Co]Cbl) bound to human transcobalamin II (TC II) were studied in HepG2 cells, a line of hepatocytes derived from a human hepatoma. The cells bound the TC II-Cbl by specific, high affinity receptors. Within the cell, the CN-Cbl was promptly freed from TC II and the CN-Cbl converted to more active forms including adenosyl Cbl (AdoCbl) and methyl Cbl (MeCbl). Whereas free labeled Cbl was still present at 72 hours after entry, the cells also bound Cbl to an intracellular binder (ICB) presumed to represent the holo enzymes dependent on Cbl. At levels of TC II that saturated the receptors for TC II-Cbl, much of the Cbl entering the cells remained free and was converted to AdoCbl. Under these circumstances the cells released free Cbl, mostly AdoCbl. Human R type binders of Cbl, which are glycoproteins and some having a terminal galactose, were bound by the HepG2 cells. The binding was characteristic of the receptor system responsive to a terminal galactose, or asialoglycoproteins, but was inconsistent and of low affinity. Cbl bound to R binder was internalized and converted to coenzyme forms of Cbl, but the process was much less effective than when the Cbl entered via the TC II receptor system. It was concluded that the receptors for R-Cbl were unlikely to contribute to the physiologic transport of Cbl in man, but may function in some yet unknown way.

Asialoglycoprotein Receptor↗
PubMed1984

The availability of therapeutic hydroxocobalamin to cells.

Hydroxocobalamin (OH-Cbl), when used to treat vitamin B12 deficiency, is better retained by the body than is cyanocobalamin (CN-Cbl), but the availability to cells has not been studied systematically. In a series of experiments, we compared the uptake and internalization of OH-Cbl and CN-Cbl bound to transcobalamin II (TCII) by a human cell model, the HeLa cell. TCII-OH-Cbl was: (1) taken up in larger amounts per unit time, (2) the greater uptake was not a consequence of more effective attachment to receptors of TCII-Cbl nor to a more rapid regeneration of receptors, (3) the difference was expressed during the phase of internalization of TCII-Cbl, (4) with CN-Cbl, the stages of binding to receptors plus internalization were more readily reversed, and (5) larger amounts of OH-Cbl were internalized and converted to active coenzyme forms of Cbl. When injected into a healthy person, 200 micrograms of OH-Cbl was better retained in the circulation than 200 micrograms of CN-Cbl. When added in vitro in equivalent amounts, more OH-Cbl was bound to nonspecific plasma proteins. This greater and broader binding neither enhanced nor interfered with the uptake of Cbl by cells, which was determined by the amount of Cbl bound physiologically to TCII. It was concluded that OH-Cbl is a more efficient form of treatment of the common types of Cbl deficiency, principally because of the better retention, which requires less frequent injections, but also because of greater availability to cells.

Animals↗
PubMed1983

Hydrophobic interactions of transcobalamin II (TC II) from mammalian sera.

The hydrophobic properties of mammalian transcobalamin IIs (TC II) were studied by chromatography of radioactive cyanocobalamin (CN[57Co]Cbl)-labeled serum on phenyl-Sepharose CL-4B. Mammalian holo TC IIs (CN[57Co]Cbl-TC II) exhibited species variability in their affinity for the hydrophobic matrix in the order: dog greater than mouse greater than human greater than rat greater than rabbit. Phenyl-Sepharose chromatography of the isolated CN[57Co]Cbl-TC II peaks from gel filtration of dog and rat serum showed no hydrophobic change in dog TC II, but an increase in hydrophobicity of rat TC II. Phenyl-Sepharose chromatography of CN[57Co]Cbl-labeled rabbit serum (holo TC II) and the unlabeled serum (apo TC II) showed apo TC II to be more hydrophobic than holo TC II as has been shown for human TC II (Begley et al., Biochem Biophys Res Commun 103:434-441, 1981). Thus mammalian holo TC IIs differ in their hydrophobic properties and apo TC II, in man and rabbit, is more hydrophobic than holo TC II. In addition, isolation of the TC II in some animal sera by gel filtration may result in a TC II that is more hydrophobic than the native molecule.

Animals↗
PubMed1982

Atypical cobalamin binding in the serum of congenital deficiency of transcobalamin II.

The serum cobalamin (Cb 1) binding patterns were described in nine children with congenital deficiency of transcobalamin II (TC II). Immunoreactive TC II was less than 100 pg/ml TC II-Cb 1 equivalent in eight and 150 pg/ml in the ninth. There was neither endogenous TC II-Cb 1 (holo TC II) nor apo TC II. Thus, the defect was characterized by the absence of any binding of Cb 1 to TC II either in vivo or in vitro and either non-detectable or much reduced immunoreactive TC II. Only the serum from an untreated infant bound any added Cb 1 at all, but in every sera there was binding of endogenous Cb 1 to a substance of the molecular size of albumin. Native Cb 1 was also bound to R binder and in some instances was incorporated into large complexes. The precise nature, cause and consequences of this atypical binding are unknown.

Adolescent↗
PubMed1981

The content, binding, and forms of vitamin B12 in milk.

The vitamin B12 (cobalamin, Cbl) content of 19 human milks ranged from 0.33 to 3.20 ng/ml, mean 0.97 ng/ml. The milk content of 10 mothers taking 5 to 100 micrograms of supplemental cyanocobalamin daily was not significantly different from that of unsupplemented mothers. The Cbl native to milk was bound to an R type binder. The R binder was also the dominant, by far, unsaturated Cbl binder, but transcobalamin II was found in every milk. The amounts of transcobalamin II were of the same order of magnitude as in serum and seemed to increase with the interval postpartum. Methylcobalamin was the most abundant Cbl of milk. Human milk from well fed mothers contains adequate amounts of Cbl, but the Cbl may become available only if there are sufficient proteolytic enzymes to release it from binding to R binder.

Chromatography, Gel↗
PubMed1981

Forms of vitamin B12 in radioisotope dilution assays.

Since the presence of analogues of vitamin B12 (B12, cobalamin, Cbl) has been postulated as the basis for the high values obtained by some radioisotope dilution assays (RIDA) of serum Cbl we examined serum for analogues. None could be demonstrated in the extracts of serum prepared for RIDA as sought by both direct and indirect techniques. The natural forms of serum Cbl were converted to cyanocobalamin (CN Cbl) by this process of extraction which included cyanide (CN). The correctly performed RIDA for Cbl based on R binder gave higher values than a RIDA based on intrinsic factor or than by bioassay. By exclusion, the difference appeared to be due to unidentified factors rather than the presence of analogues.

Chromatography, Gel↗