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Biomedical subjects

J M Scott

Publications and source records attributed to J M Scott.

At least 19 recordsLinked to original sources
PubMed1992

The relationship between the activity of methionine synthase and the ratio of S-adenosylmethionine to S-adenosylhomocysteine in the brain and other tissues of the pig.

Using nitrous oxide to inactivate methionine synthase in vivo, the relationship of the activity of methionine synthase to the S-adenosylmethionine (AdoMet)/S-adenosylhomocysteine (AdoHcy) ratio was examined in neural and other tissues of the pig. Pigs were exposed to 15% nitrous oxide for varying intervals of up to 7 days or studied at varying intervals of recovery in air after 7 days nitrous oxide inhalation, and the rate of inactivation or resynthesis of methionine synthase was related to the corresponding AdoMet/AdoHcy ratios. The rate of inactivation of enzyme during nitrous oxide exposure was considerably faster in the liver and kidney than in the brain and spinal cord with activity levelling off between 10% and 20% of control values. The AdoMet/AdoHcy ratio fell in all tissues during nitrous oxide treatment, the fall being most marked in the brain and spinal cord where a 10-fold change occurred. This change was attributed mainly to a rise in AdoHcy levels. The recovery pattern of methionine synthase was broadly linear but was slower in the spinal cord (0.10 +/- 0.03% per hr; mean +/- SEM) than in any other tissue examined including brain (0.35 +/- 0.04% per hr). Correspondingly, the recovery of the AdoMet/AdoHcy ratio was also significantly slower in the spinal cord. When values for exposure and recovery were combined there was a significant correlation between the activity of methionine synthase and the AdoMet/AdoHcy ratio in both the brain (r = 0.90; P < 0.001) and the spinal cord (r = 0.92; P < 0.001). These results support the concept that the AdoMet/AdoHcy ratio is closely related to the pathogenic process which produces the neurologic lesions associated with a reduction in methionine synthase activity.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1992

Choline transport into rat liver mitochondria. Characterization and kinetics of a specific transporter.

Rat liver mitochondria possess a specific choline transporter in the inner membrane. The transporter shows saturable kinetics at high membrane potential with a Km of 220 microM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 degrees C. At physiological concentrations of choline, the rate of choline uptake by the transporter shows a linear dependence on membrane potential; uptake is distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all analogues of choline, are high affinity competitive inhibitors of choline transport with Ki values of 17, 55, 15, and 127 microM, respectively. The choline transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. Analogue studies have shown that the hydroxyl and the quaternary ammonium groups of choline are necessary for binding to the transporter. A comparison of molecular models of choline and the high affinity inhibitors has provided evidence for the preferred conformation of choline for binding to the transporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.

Animals↗
PubMed1992

Correlation of the ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine in the brain and cerebrospinal fluid of the pig: implications for the determination of this methylation ratio in human brain.

1. Pigs were maintained in air or in an atmosphere of nitrous oxide which dramatically changes the S-adenosyl-L-methionine to S-adenosyl-L-homocysteine ratio in neural tissues. Samples of cerebrospinal fluid, cortex, cerebellum and spinal cord were then extracted and analysed for S-adenosyl-L-methionine and S-adenosyl-L-homocysteine. Regression analyses were carried out on values obtained in cerebrospinal fluid and in neural tissues. 2. Highly significant correlations were obtained between levels of S-adenosyl-L-homocysteine (r2 = 0.42-0.69; P less than 0.001) and S-adenosyl-L-methionine/S-adenosyl-L-homocysteine ratios (r2 = 0.56-0.65; P less than 0.001) in cerebrospinal fluid and levels and ratios in cortex, cerebellum and spinal cord. The levels of S-adenosyl-L-methionine did not show a significant correlation. 3. We conclude that the ratio of these metabolites in the cerebrospinal fluid may reflect the ratio in the central nervous system and we suggest that this may also be true in human tissues. This finding will permit the determination of the probable methylation ratio in the central nervous system in human conditions, such as vitamin B12 deficiency and acquired immune deficiency syndrome, where a similar myelopathy occurs to that seen in the nitrous oxide-treated pig. All three myelopathies may arise from an inhibition of methyltransferases involved in the synthesis of myelin that would occur when the methylation ratio is reduced.

Animals↗
PubMed1992

Cobalt-vitamin B-12 deficiency decreases methionine synthase activity and phospholipid methylation in sheep.

Two groups of lambs were fed either a Co-deficient or a Co-sufficient whole barley-based diet for 28 wk to induce a severe Co-vitamin B-12 deficiency. Holo and apo methionine synthase activities were significantly lower in the liver, kidney and spinal cord of Co-deficient animals compared with controls. Neither form of this enzyme in the brain was affected by Co deficiency. The ratio of the tissue concentrations of S-adenosyl methionine to S-adenosyl homocysteine was significantly lower only in the liver of Co-deficient animals, suggesting that the activity of hepatic SAM-dependent methyltransferase enzymes would be impaired. Measurements of tissue concentrations of phosphatidyl choline and phosphatidyl ethanolamine revealed lower concentrations of phosphatidyl choline and a lower phosphatidyl choline:phosphatidyl ethanolamine ratio in both liver and brain of the Co-deficient animals. The latter finding occurred in the absence of changes in either methionine synthase activity or the methylation ratio and may result from impaired availability of hepatic phosphatidyl choline for transport into the brain.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1992

Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

Cell Line↗
PubMed1991

Evidence of brain methyltransferase inhibition and early brain involvement in HIV-positive patients.

The myelopathy associated with human immunodeficiency virus (HIV) infection closely resembles that in subacute combined degeneration, a disorder of vitamin B12 metabolism. To investigate whether the disorders share a pathogenetic mechanism, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were measured in the cerebrospinal fluid (CSF) of 20 HIV-seropositive patients and 30 HIV-negative patients who were undergoing lumbar puncture for other medical reasons. The HIV-seropositive patients had significantly lower CSF concentrations of SAM (mean 77 [SD 25] vs 131 [35] nmol/l; p less than 0.001) and significantly higher concentrations of SAH (30.5 [6.8] vs 19.0 [7.1] nmol/l; p less than 0.001) than the controls. There was therefore a significant difference between the groups in the SAM/SAH (methylation) ratio (HIV 2.7 [1.0] vs control 7.6 [3.4]; p less than 0.001). There were no correlations between SAM or SAH concentrations or methylation ratio and age or sex in both groups, or serum B12 and folate concentrations, CSF folate, serum or CSF methylmalonic acid, risk factors, body mass index, specific drug treatment received, or disease stage in the HIV group. This finding suggests that HIV affects the brain from a very early stage of the infection. We suggest that, as in the pig, the CSF methylation ratio closely reflects that in the brain. In HIV-infected patients a reduced brain methylation ratio would inhibit methyltransferase enzymes, which would lead to hypomethylation in the central nervous system and ultimately to neurological lesions. In a pig model of subacute combined degeneration and in vitamin-B12-deficient human beings, the primary cause of the low methylation ratio is impaired recycling of SAH back to SAM, a process which requires vitamin-B12-dependent methionine synthase. The HIV patients in this study were vitamin B12 and folate replete, which suggests a different cause for the low methylation ratio.

Adult↗
PubMed1991

Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses.

To investigate human immune responses to defined leishmania Ag we have begun to characterize biochemically and immunologically, an abundant 42-kDa surface Ag of Leishmania amazonensis, a causative agent of human leishmaniasis. We have shown that this Ag, La gp42, is expressed on the surface of L. amazonensis promastigotes, being anchored to the membrane by a glycosyl-phosphatidylinositol moiety. As demonstrated by lectin blotting studies, La gp42 is glycosylated, binding both Con A and wheat germ agglutinin. Immunologically, La gp42 is strongly recognized by sera from patients with different forms of leishmaniasis as well as by patients with Chagas' disease. In addition, we show that purified La gp42 stimulates the proliferation of human T lymphocytes obtained from several leishmaniasis patients. Finally, the N-terminal sequence of La gp42 was obtained and a serologically cross-reactive 42-kDa protein with a homologous sequence was identified in Leishmania major.

Amino Acid Sequence↗
PubMed1991

Betaine-homocysteine methyltransferase: organ distribution in man, pig and rat and subcellular distribution in the rat.

1. Conflicting reports exist as to the organ distribution of betaine-homocysteine methyltransferase (EC 2.1.1.5). It is important to establish its presence or absence in brain, since its substrate, betaine, has recently become established in the treatment of certain diseases involving this organ. 2. It remains unclear whether the reported success of this treatment results from the use of betaine to methylate homocysteine and produce methionine in situ in neural tissue, or whether the effect is secondary to these same reductions happening in other organs, such as the liver. The former would require the presence of betaine-homocysteine methyltransferase in neural tissue. 3. This study demonstrates the complete absence of any activity for this enzyme in the brain of the three species examined. The enzyme was found to be present in both the liver and kidney of man and pig, but only in the liver of the rat. 4. The only source of betaine in cells is via the oxidation of choline. Since the enzymes involved in this conversion have never been shown to exist anywhere other than the mitochondria, it has been assumed that the methyltransferase is also mitochondrial. In this study, it is demonstrated that the enzyme exists only in the cytoplasm of rat liver cells.

Animals↗
PubMed1991

A rapid and simple diagnostic test for active visceral leishmaniasis.

We have developed an immunodot assay for the serodiagnosis of active visceral leishmaniasis (AVL) which utilizes protein A colloidal gold as the visualizing agent. The test is simple, requires few reagents, and can be completed in two hours. It is sensitive and specific for active visceral leishmaniasis, and generally correlates with the ELISA. Either whole blood or sera in minute quantities may be used as test antibody. In addition, the use of the protein A gold immunodot is shown to detect anti-leishmania antibodies in infected dogs.

Animals↗
PubMed1990

A new high performance liquid chromatographic method for the simultaneous measurement of S-adenosylmethionine and S-adenosylhomocysteine. Concentrations in pig tissues after inactivation of methionine synthase by nitrous oxide.

A rapid gradient method has been established to measure simultaneously the tissue levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). The method involves application of a 15%-25% linear gradient of methanol over a period of 10 min to an initial mobile phase of 15% methanol with 25 mM sodium phosphate, 10 mM 1-heptanesulfonic acid solution at pH 3.2 and a flow rate of 1 mL/min. AdoHcy elutes at 9.5 min and AdoMet at 12.6 min. The assay has a detection limit of 10 pmol and is linear in the concentration range 30-800 pmol. The method was used to assess changes in AdoMet and AdoHcy concentrations of pig tissues after seven days exposure to the anaesthetic gas nitrous oxide which irreversibly inactivates methionine synthase and induces an inability to recycle homocysteine, particularly in neural tissues. The treatment caused significant alterations in cellular AdoMet:AdoHcy ratios which were principally due to a dramatic rise in AdoHcy concentrations.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1990

The value of the erythrocyte indices as a screening procedure in predicting nutritional deficiencies.

The results of a large number of nutritional screen requests (n = 871) were compared with corresponding values of erythrocyte indices considered predictive of nutritional deficiencies to determine if such indices could be used in a prospective screening procedure to restrict the number of serum vitamin B12, folate, and ferritin assays. Low mean cell haemoglobins (MCH less than 27 pg) were found to be superior to low mean cell volumes (MCV less than 77 fl), in predicting low serum ferritin values. The occurrence of deficient ferritin values was 90% when the MCH was very low (MCH less than 23 pg). Vitamin B12 or folate deficiency could not be predicted from the MCV. A normal MCV was found in more than 55% of vitamin B12 deficient samples and some 30% of serum B12 deficients (less than 150 micrograms/l) showed no evidence of anaemia (Hb greater than 12 gm/dl) or macrocytosis (MCV less than 100 fl). It would not seem appropriate to use erythrocyte indices alone as a method of selecting samples for further investigation of folate or vitamin B12 status.

Adult↗
PubMed1990

Use of beta-lactamase to hydrolyse interfering antibiotics in vitamin B12 microbiological assay using Lactobacillus leichmannii.

The presence of certain antibiotics in patients' sera can invalidate microbiological assays for vitamin B12 using Lactobacillus leichmannii by inhibiting organism growth. Analysis of 3291 consecutive serum vitamin B12 assays in a large general hospital service showed inhibition of growth in 303 samples (9.2%). In nearly all cases the interfering substance was a beta-lactam antibiotic. The value of using a beta-lactamase preparation to eliminate antibiotic effects was studied using antibiotic-containing patient sera and also normal serum to which antibiotics were added. In-vitro additions of penicillins, cephalosporins, and other antibiotics to normal serum resulted in varying levels of organism inhibition, but excellent recovery of a control value after treatment with beta-lactamase (greater than 98%). Patient sera showing inhibition were reassayed in duplicate following treatment with beta-lactamase (n = 200), the overall effectiveness of the treatment being verified by the excellent recovery (greater than 98%) of a cyanocobalamin 'spike' added to a control. In only 0.2% of cases (6 of 3291 samples) was this treatment ineffective and it has thus proved an efficient method of eliminating the problem of antibiotic interference in the vitamin B12 microbiological assay.

Anti-Bacterial Agents↗
PubMed1990

In vitro responses to Leishmania antigens by lymphocytes from patients with leishmaniasis or Chagas' disease.

T cell responses are correlated with recovery from and resistance to leishmaniasis. Antigens of Leishmania chagasi were evaluated by determining their ability to elicit in vitro proliferation and cytokine production in peripheral blood lymphocytes and in T cell lines and clones from patients with histories of leishmaniasis or Chagas' disease. Antigens tested were selected by their reactivity with patient antibodies. Several of the antigens induced proliferative responses in peripheral blood lymphocytes from patients recovered from visceral or cutaneous leishmaniasis or with chronic Chagas' disease. Two purified glycoproteins, 30 and 42 kD, were consistently among the most effective in eliciting high proliferative responses and IL-2 production. Lymphocytes from a recovered visceral leishmaniasis patient were used to produce T cell lines against either the 30- or 42-kD antigen. Each of the lines responded to both of these antigens as well as to crude leishmania lysate. CD4+ T cell clones specific for either or both of these antigens were also isolated from a visceral leishmaniasis patient. In contrast, rabbit antisera produced against these two antigens were not crossreactive. Both antigens were effective in inducing the production of IFN-gamma from T cell lines from both leishmaniasis and Chagas' disease patients. These studies demonstrate the potential for defining parasite antigens with broad immunostimulatory capabilities.

Animals↗