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R H Allen

Publications and source records attributed to R H Allen.

At least 91 records · Page 5Linked to original sources
PubMed1989

Structure of the cDNA encoding transcobalamin I, a neutrophil granule protein.

Transcobalamin I (TCI) is a member of the R binder family of vitamin B12 binding proteins. It is a major protein constituent of secondary granules in neutrophils. We have isolated and characterized full length cDNA clones encoding TCI in order to determine whether its expression is coordinately regulated with the appearance of secondary granules and whether it is consequently a useful marker of granulocyte development. Partial amino acid sequences of human R protein were obtained from tryptic digestion fragments. Using the polymerase chain reaction, a partial TCI cDNA probe was isolated by selective amplification of a region of cDNA located between two oligonucleotides deduced from the available partial amino acid sequences. The amplified probe was then used to obtain full length clones from a granulocyte cDNA library. Identity of the clones was confirmed by matching DNA sequence to known peptide amino acid sequence. TCI is transcribed to a single 1.5-kilobase mRNA species. The predicted protein sequence is 433 amino acids long. We have compared the sequence of TCI to that of rat intrinsic factor. The two proteins have areas of extensive homology which implicate regions potentially important for vitamin B12 binding. TCI mRNA was present in late neutrophil precursors but absent from uninduced and induced HL60 cells.

Amino Acid Sequence↗
PubMed1989

Effect of betahistine dihydrochloride compared with cinnarizine on induced vestibular nystagmus.

The effect of betahistine compared with cinnarizine on induced vestibular nystagmus was evaluated using a rotating chair, in 6 healthy volunteers. The subjects underwent a slow acceleration followed by a sudden stop. Electronystagmograph tracings were taken initially as pretreatment control values, and after betahistine 8 mg t.i.d. and cinnarizine 15 mg t.i.d. had been taken. The duration of nystagmus and average eye speed were measured. No difference was recorded in either parameter between the pretreatment rotation and that following betahistine (P greater than 0.05). A significant difference (P less than 0.05) was seen in the duration of nystagmus during initial acceleration, and in average eye speed following the sudden stop after treatment with cinnarizine.

Adult↗
PubMed1988

Determination of the carbohydrate composition of mammalian glycoproteins by capillary gas chromatography/mass spectrometry.

A technique to determine the carbohydrate composition of glycoproteins using capillary gas chromatography/mass spectrometry (electron impact) with selected ion monitoring is described. This method entails hydrolysis with methanolic-HCl followed by formation of trimethylsilyl methylglycoside derivatives, extraction of the carbohydrate derivatives into hexane, and GC/MS analysis. For those carbohydrates that are present in animal glycoproteins including fucose, mannose, galactose, glucosamine, galactosamine, and N-acetylneuraminic acid (sialic acid), the sensitivity of this assay was approximately 1-3 pmol and the assay was linear over a 100-fold range. The carbohydrate compositions determined on small quantities (1-10 pmol) of various glycoproteins including human transferrin and alpha-1 acid glycoprotein, fetuin, and ovalbumin were identical to their reported carbohydrate content and compositions. Major advantages of this technique include the time required to complete the sample preparation and analysis (less than 8 h), the sensitivity and specificity of the assay, and the fact that all carbohydrate moieties, including sialic acid, can be quantitated in a single hydrolysate of a glycoprotein.

Carbohydrates↗
PubMed1988

Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis.

Among 141 consecutive patients with neuro-psychiatric abnormalities due to cobalamin deficiency, we found that 40 (28 percent) had no anemia or macrocytosis. The hematocrit was normal in 34, the mean cell volume was normal in 25, and both tests were normal in 19. Characteristic features in such patients included paresthesia, sensory loss, ataxia, dementia, and psychiatric disorders; longstanding neurologic symptoms without anemia; normal white-cell and platelet counts and serum bilirubin and lactate dehydrogenase levels; and markedly elevated serum concentrations of methylmalonic acid and total homocysteine. Serum cobalamin levels were above 150 pmol per liter (200 pg per milliliter) in 2 patients, between 75 and 150 pmol per liter (100 and 200 pg per milliliter) in 16, and below 75 pmol per liter (100 pg per milliliter) in only 22. Except for one patient who died during the first week of treatment, every patient in this group benefited from cobalamin therapy. Responses included improvement in neuropsychiatric abnormalities (39 of 39), improvement (often within the normal range) in one or more hematologic findings (36 of 39), and a decrease of more than 50 percent in levels of serum methylmalonic acid, total homocysteine, or both (31 of 31). We conclude that neuropsychiatric disorders due to cobalamin deficiency occur commonly in the absence of anemia or an elevated mean cell volume and that measurements of serum methylmalonic acid and total homocysteine both before and after treatment are useful in the diagnosis of these patients.

Adolescent↗
PubMed1988

Failure to detect beta-leucine in human blood or leucine 2,3-aminomutase in rat liver using capillary gas chromatography-mass spectrometry.

It has been reported (Poston, J. (1976) J. Biol. Chem. 251, 1859-1863; (1982) 255, 10067-10072; (1984) 259, 2059-2061) that mammalian tissues contain an adenosylcobalamin-dependent enzyme, leucine 2,3-aminomutase, which catalyzes the interconversion of beta-leucine and leucine. It was also reported that beta-leucine is detectable in normal human serum (mean = 4.8 mumol/liter, n = 37) and is elevated in serum from patients with cobalamin deficiency (mean = 24.7 mumol/liter, n = 17). Serum levels of leucine were claimed to be decreased in the cobalamin deficient patients (mean = 52 mumol/liter) as compared with the normal subjects (mean = 81 mumol/liter). It was also reported that rat liver supernatant catalyzed the formation of beta-leucine, leucine, or both amino acids from iso-fatty acids, and that the generation of leucine from iso-fatty acids was stimulated by adenosylcobalamin and inhibited by unsaturated cobalamin-binding protein. We have synthesized t-butyldimethylsilyl derivatives of beta-leucine and leucine and have used capillary gas chromatography-mass spectrometry for their analysis. Using forms of beta-leucine and leucine that contain several deuterium atoms in place of several hydrogen atoms as internal standards, techniques have been developed which make it possible to detect and quantitate as little as 0.1 mumol/liter of beta-leucine or leucine in human serum and in incubations containing rat liver supernatant. beta-Leucine was not detectable, i.e. less than 0.1 mumol/liter, in any sera from 50 normal human subjects or in any sera from 50 cobalamin-deficient patients. The mean level of leucine in the 50 cobalamin-deficient sera was 219 mumol/liter, which was not decreased with respect to that in the 50 control sera (167 mumol/liter). Experiments in which beta-leucine, leucine, isostearic acid, or isocaproic acid were incubated with rat liver supernatant in the presence or absence of adenosylcobalamin or cobalamin-binding protein failed to demonstrate the formation of leucine or beta-leucine or their interconversion under any of the conditions studied. We conclude that beta-leucine is not present in human blood and that the existence of leucine 2,3-aminomutase in mammalian tissues remains to be established.

Adult↗
PubMed1988

Therapeutic approaches to cobalamin-C methylmalonic acidemia and homocystinuria.

The use of hydroxocobalamin (OH-B12), betaine, carnitine, and folinic acid were studied in two children with the cobalamin C form of methylmalonic acidemia and homocystinuria. When daily injections of 1 mg OH-B12 were discontinued for 3 weeks, there was no significant change in total plasma homocysteine or methionine levels and only a modest increase in methylmalonate. Orally administered OH-B12 1 mg/d in one patient was associated with an increase in plasma homocystine and a decrease in methionine within 1 month. Withdrawal of betaine 250 mg/kg/d was also associated with a rise in plasma homocystine and a fall in methionine levels. Carnitine 100 mg/kg/d lead to an increase in urinary excretion of propionylcarnitine, but did not affect plasma methylmalonate levels. No beneficial biochemical effect of folinic acid could be documented at a dose of 25 mg/d. Our results suggest that daily injections of OH-B12 are not necessary to maintain metabolic control and that orally administered OH-B12 is unlikely to be effective. Betaine appears to act synergistically with OH-B12 and should be part of the treatment regimen. Although there are theoretical reasons for using L-carnitine and folinic acid, we could not document their effectiveness in these two patients.

Administration, Oral↗
PubMed1988

Molecular cloning of L-methylmalonyl-CoA mutase: gene transfer and analysis of mut cell lines.

L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency.

Animals↗
PubMed1988

Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry.

To determine if levels of serum total homocysteine are elevated in patients with either cobalamin or folate deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure total homocysteine in the serum of 78 patients with clinically confirmed cobalamin deficiency and 19 patients with clinically confirmed folate deficiency. Values ranged from 11 to 476 mumol/liter in the cobalamin-deficient patients and 77 of the 78 patients had values above the normal range of 7-22 mumol/liter as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum total homocysteine was positively correlated with serum folate, mean corpuscular volume, serum lactate dehydrogenase, serum methylmalonic acid, and the degree of neurologic involvement, and inversely correlated with platelets and hematocrit. In the folate-deficient patients, values for serum total homocysteine ranged from 17 to 185 mumol/liter and 18 of the 19 patients had values above the normal range. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of total homocysteine while they were free of hematologic and neurologic abnormalities. The measurement of serum total homocysteine will help define the incidence of cobalamin deficiency and folate deficiency in various patient populations.

Adult↗
PubMed1987

Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry.

Total homocysteine, total cysteine, and methionine have been extracted and partially purified from serum and urine using reduction with 2-mercaptoethanol followed by cation-exchange chromatography and anion-exchange chromatography. The t-butyldimethylsilyl derivatives were prepared and analyzed using capillary gas chromatography-mass spectrometry with selected ion monitoring. The addition of DL-[3,3,3',3',4,4,4',4'-2H8]homocystine, DL-[3,3,3',3'-2H4]cystine, and L-[methyl-2H3]methionine to the starting samples prior to the reduction of all disulfides, including the deuterated internal standards, with 2-mercaptoethanol makes it possible to quantitate all three amino acids. Normal ranges for total homocysteine, total cysteine, and methionine have been determined in human and rat serum and in human urine.

Amino Acids↗
PubMed1986

Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatography-mass spectrometry.

To determine the incidence of elevated levels of serum methylmalonic acid in patients with cobalamin deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure methylmalonic acid in the serum of 73 patients with clinically confirmed cobalamin deficiency. Values ranged from 55 to 22,300 ng/ml, and 69 of the 73 patients had values above the normal range of 19-76 ng/ml as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum methylmalonic acid was significantly correlated with the serum folate level and the degree of neurologic involvement. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of methylmalonic acid while free of hematologic and neurologic abnormalities. A cobalamin-deficient patient is described with a normal serum cobalamin and an elevated serum methylmalonic acid. We conclude that the ability to measure methylmalonic acid in human serum will be useful in studies designed to determine the incidence of cobalamin deficiency in various patient populations.

Adolescent↗
PubMed1985

Isolation and characterization of methionine synthetase from human placenta.

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗