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H N Munro

Publications and source records attributed to H N Munro.

At least 37 records · Page 2Linked to original sources
PubMed1987

Glycoconjugates as noninvasive probes of intrahepatic metabolism: I. Kinetics of label incorporation with evidence of a common precursor UDP-glucose pool for secreted glycoconjugates.

Conditions are described under which hepatic metabolism of administered labeled sugars can be monitored in the intact rat by measuring output of label from the liver in two secreted glycoconjugates derived from intracellular UDP-glucose, namely glucuronic acid linked to an administered drug (acetaminophen), and galactose secreted in glycoproteins. Rats with indwelling intrajugular catheters were given constant infusions of acetaminophen at nontoxic doses, and acetaminophen-glucuronide was isolated from rat urine by HPLC. When an unprimed continuous infusion of [U-14C]glucose or [1-3H]galactose is begun during acetaminophen infusion, labeling of urinary acetaminophen-glucuronide attains a plateau with an apparent half-life of 2.1 h from labeled glucose as the 14C-donor, and 1.2 h from labeled galactose. The times required to attain steady-state are attributable either to formation or to hepatic secretion of the glucuronyl conjugate rather than to mixing in extracellular pools or to delays in renal excretion since the half-time of urinary excretion of intravenously injected acetaminophen-glucuronide is only 21 minutes. Consequently, the excreted glucuronide provides noninvasive evidence of the recent specific activity in the precursor pool of hepatic UDP-glucose feeding glucuronic acid. The second glycoconjugate used for comparison was galactose, isolated from the acute-phase plasma protein alpha 1-acid glycoprotein. This represents hepatic UDP-galactose labeling. Comparison of the labeling patterns of secreted galactose and glucuronic acid demonstrates that they are derived from the same intracellular pool of UDP-glucose. This is in contrast to the patterns of labeling of hepatic glycogen from (1-3H) and [U-14C]glucose which have been shown to diverge from those of secreted glucuronic acid.

Acetaminophen↗
PubMed1987

Glycoconjugates as noninvasive probes of intrahepatic metabolism: II. Application to measurement of plasma alpha 1-acid glycoprotein turnover during inflammation.

We describe a new method for measuring plasma glycoprotein turnover in vivo using carbohydrate-moiety labeling compared with precursor UDP-sugar monitoring, which allows estimation of the specific activity of the precursor at the biosynthetic site of the glycoprotein. This method has been applied to plasma alpha 1-acid glycoprotein (AGP) kinetics, and has allowed direct quantitation of absolute synthesis rates of AGP in a non-steady state. Following turpentine-induced acute inflammation, AGP was found to undergo a maximum increase in plasma level of eight-fold with a 20- to 25-fold induction in absolute synthesis rate peaking at 25 to 50 h, and a concurrent 2.0 to 2.5-fold increase in fractional degradation rate. The changes in absolute synthesis rates were quite comparable both temporally and quantitatively to changes in hepatic AGP mRNA levels and gene transcription rates reported by others following turpentine inflammation, thus suggesting that AGP synthesis in vivo is predominantly regulated by the level of its mRNA. The carbohydrate moiety labeling method can be applied to other plasma glycoproteins to measure their kinetic parameters in the intact animal or human subject.

Acetaminophen↗
PubMed1987

Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression.

Ferritin stores iron within a protein shell consisting of 24 subunits of two types, heavy (H) and light (L). According to Southern blotting, the rat genome contains four copies homologous to the H-subunit cDNA (H cDNA). To determine whether only one of these is expressed, H cDNAs isolated from rat liver and heart mRNAs were compared and found to share identical nucleotide sequences. Next, genomic clones for three of the four rat H-subunit loci were isolated. Two were classical processed pseudogenes, whereas the third contained an expressed gene. RNase intron mapping of this expressed gene generated the same exon protection pattern when total RNA from rat liver or heart was used, indicating that this gene accounts for most or all of the H-subunit mRNAs (H mRNAs) in these tissues. Comparison of the expressed rat H-subunit gene (H gene) structure with published sequences for other species displays considerable conservation. The coding sequence of the rat H gene predicts 95% similarity to the human amino acid sequence, thus being more highly conserved than the L-subunit sequence of these species. Near the cap region of the 5' untranslated region, the rat H mRNA displays a 28-nucleotide sequence that is almost totally conserved in the corresponding region of the human, bullfrog, and chicken H mRNA and is also faithfully represented in the rat and human L-subunit mRNAs (L mRNAs), thus making this sequence a prime candidate for involvement in the known translational regulation of both subunits by iron. In the 5' flanking region, partially conserved sequences common to H gene and L-subunit gene (L gene) of the rat may be involved in transcriptional regulation by iron, whereas those conserved only in the H gene of man and the rat imply that other factors may independently control H-subunit regulation.

Amino Acid Sequence↗
PubMed1987

Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region.

In previous studies, we showed that acute administration of iron to intact rats or to rat hepatoma cells in culture induces synthesis of the iron-storage protein ferritin by activating translation of inactive cytoplasmic ferritin mRNAs for both the heavy (H) and the light (L) subunits. In the course of activation, these ferritin mRNAs are recruited onto polysomes. To elucidate the structural features of these mRNAs involved in the translational response to iron, a chimera was constructed from the 5' and 3' untranslated regions (UTRs) of ferritin L subunit mRNA fused to the reading frame of the mRNA of bacterial chloramphenicol acetyltransferase (CAT). This chimera and deletion constructs derived from it were introduced into a rat hepatoma cell line by retrovirus-mediated gene transfer. The complete chimera showed increased CAT activity in response to iron enrichment of the medium, whereas deletion of the first 67 nucleotides of the 5' UTR, which contain a highly conserved sequence, caused loss of regulation by iron. Whereas cis-acting sequences located in the 5' flanking regions of many genes have been repeatedly implicated in modulating their transcriptional expression, we report here a specific regulatory translational sequence found within the 5' UTR of a eukaryotic mRNA.

Animals↗
PubMed1987

Protein nutriture of a group of free-living elderly.

The adequacy of the protein intakes of elderly people without overt debilitating diseases was investigated on 691 free-living men and women divided into those aged 60-75 y and those greater than 75 y. In both age groups men and women had average protein intakes of 1.02-1.06 g/kg body weight, values well above the safe level of 0.75 g/kg recommended in a WHO/FAO/UNU report. Although plasma concentrations of albumin, prealbumin, and transferrin declined with age, these were not related to low intakes of protein by individual elderly people. Similarly, upper-arm muscle mass was not less in those elderly people at the lower end of the range of protein intakes. Thus in this population of overtly healthy elderly men and women, there was no evidence of protein deficiency in contrast to other surveys where elderly people with chronic diseases were included.

Age Factors↗
PubMed1986

Both subunits of rat liver ferritin are regulated at a translational level by iron induction.

A few hours after administering iron to rats, liver ferritin synthesis increases several fold. However, Northern blot analysis with cDNA probes for ferritin light (L) and heavy (H) subunit mRNAs failed to show an increase in total population of either messenger. Cytoplasmic distribution of ferritin messages was therefore investigated in control and iron administered rats killed at 3.5 hours. The liver post-mitochondrial supernatant was fractionated on a sucrose gradient to separate polyribosomes, monosomes, ribosomal subunits and cell sap. RNA extracted from each fraction and analyzed using Northern blotting showed that 65% of the total mRNA population for each subunit was present in the cell sap of control rats, presumably as mRNP particles since ribosomal RNA was absent from this fraction. After iron administration, these reserves of free mRNA were recruited onto the polysomes, reducing the free mRNA pool to 15% of the total. We interpret this to be due to activation of blocked ferritin messages on entry of iron into the cell.

Animals↗
PubMed1986

Glycoconjugates as noninvasive probes of intrahepatic metabolism: pathways of glucose entry into compartmentalized hepatic UDP-glucose pools during glycogen accumulation.

Recent studies have questioned the efficiency with which administered glucose generates hepatic glycogen through the direct nonrecycling route compared with the indirect route from glucose recycled through glycolysis followed by gluconeogenesis. Using fasted and refed rats, we examined the relative access of infused [1-3H]- and [U-14C]glucose by way of these two pathways to liver glycogen and to hepatic glucuronic acid, the latter recovered from the urine as the glucuronide conjugated with administered acetaminophen. In fasted animals and during early refeeding, extensive dilution of administered [3H]- and [14C]glucose recovered in glycogen showed that 60-70% of the labeled glucose had undergone recycling by the indirect route. As refeeding progressed with substantial glycogen deposition, the contribution of the recycling pathway to glycogen and glucuronic acid diminished considerably. Thus, there is a shift in pathways of hepatic glucose utilization as liver glycogen accumulates. Consequently, the ratio of 3H/14C in glucuronic acid was closely correlated with the glycogen content of the liver at sacrifice, indicating that this ratio may prove useful as a noninvasive indicator of liver glycogen concentration. Since glycogen and glucuronic acid are derived by single reactions from UDP-glucose, they should show a common labeling pattern with 3H and 14C under various nutritional conditions. However, detailed analysis of their labeling patterns showed a striking divergence, implying that there must be compartmentation of the UDP-glucose pools leading to each of these end products, either because they are made in separate compartments within the same cell or because each is made in different hepatocyte populations. We favor the former explanation because galactose secreted in glycoproteins shows 3H and 14C labeling patterns similar to those of glucuronic acid conjugated with acetaminophen, and both of these conjugations occur in the endoplasmic reticulum of the liver, whereas most glycogen is present in the cytosol.

Animals↗
PubMed1985

Isolation of alpha 1-acid glycoprotein from human plasma using high-performance liquid chromatography.

A method for the rapid isolation of purified alpha 1-acid glycoprotein (AGP) from small volumes of human plasma using HPLC has been developed. The method involves preparation of the seromucoid fraction of plasma by sequential perchloric acid and phosphotungstic acid precipitations, followed by chromatography on an HPLC TSKG-3000 column. The yield was high (0.75 mg AGP/ml plasma) and the procedure takes less than 1 day. The method lends itself to easy automation and is particularly suitable for isotopic turnover studies requiring multiple plasma samples.

Chromatography, High Pressure Liquid↗
PubMed1985

Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography.

T4 RNA ligase has been used to construct a series of defined oligoribonucleotides. Hexamer or pentamer blocks were synthesized first by multiple additions of mononucleotide diphosphates to trimers with T4 RNA ligase and removal of the terminal phosphate with alkaline phosphatase; inhibitors of the ligase were removed by passing the sample over a 1-ml reverse-phase octadecasilyl column. The two nucleotide blocks were then ligated to give undecamers. Yields for the individual ligations ranged from 85 to 100% for acceptors lacking uridines and at least 70% for those containing uridines. The overall yield of the undecamer relative to the starting trimers was about 10%. Each round of ligation averaged about 8 h; the time required to synthesize each undecamer was 1 to 2 weeks. Optimization of the steps to achieve this is described in detail.

Autoradiography↗
PubMed1985

Structure of human ferritin light subunit messenger RNA: comparison with heavy subunit message and functional implications.

Ferritin has a protein shell of 5 X 10(6) Da consisting of 24 subunits of two types, a heavier (H) chain of 21,000 Da and a lighter (L) chain of 19,000 Da. A cDNA clone of the messenger for the L subunit has been isolated from a human monocyte-like leukemia cell line. The clone contains an open reading frame of 522 nucleotides coding for an amino acid sequence matching 97% of the published sequence of human liver ferritin L subunit determined by sequenator, but it corresponds to only 55% of the reported amino acid sequence of a human liver H-subunit clone. Nevertheless, computer analysis of the subunit conformations predicted from the open reading frames of the L and H clones shows that most of the amino acid differences are conservative and would allow both subunits to form the five alpha-helices and beta-turns established by x-ray crystallography for horse spleen ferritin subunits. This suggests that L and H subunits are structurally interchangeable in forming an apoferritin shell. The 5' untranslated region of our human ferritin L clone has considerable homology with that of the rat liver ferritin L clone in the region immediately upstream from the initiator codon, notably showing an identical sequence of 10 nucleotides at the same position in both subunit clones that may participate in regulating the known activation of ferritin mRNA after iron administration. Extensive homology, including several blocks of nucleotides, was identified between the 3' untranslated regions of the human and rat L clones. The common structural features of the H and L subunits lead us to conclude that they have diverged from a single ancestral gene.

Base Sequence↗
PubMed1985

Energy intake can determine albumin synthesis in man after surgery.

Albumin synthesis rate, nitrogen balance, plasma hormone levels, and selected substrates were measured after operation in 12 patients who underwent colonic operations who were randomized to receive an intravenous fluid regimen that contained either 3.5% amino acids with 20% fat and 2.5% glucose or 3.5% amino acids with 20% fat alone. The albumin synthesis rate was higher in patients who received the first of these intravenous mixtures (357 +/- 34 mg/kg/day versus 216 +/- 22 mg/kg/day; p less than 0.01), but they also had a significantly higher intake of calories (10.2 +/- 1.1 calorie/kg/day versus 6.4 +/- 0.6 calorie/kg/day; p = 0.01). The mean albumin synthesis rate in the group who received amino acids with glucose and fat is the highest we have measured in our series of studies. Although a previous trial in a similar group of patients suggested that glucose acts on albumin synthesis by diverting uptake of amino acids into skeletal muscle, it is possible that the impressive increase in the albumin synthesis rate in patients of the present series who received supplementary glucose is related to the extra energy infused. In contrast, nitrogen balance was similar in both groups, and thus was not predictive of protein synthesis. In addition, myofibrillar protein degradation appears to be equivalent in the two groups, as indicated by 3-methylhistidine output. Plasma albumin synthesis thus may be sensitive, especially to energy intake.

Adenocarcinoma↗
PubMed1984

Conservation in rat liver of light and heavy subunit sequences of mammalian ferritin. Presence of unique octopeptide in the light subunit.

Ferritin, an iron-storage protein found in all life forms examined, is composed of varying proportions of two subunits of different molecular weight, heavy (H) and light (L). Using cDNA clones, we have determined the nucleotide sequence corresponding to the mRNA of the L-subunit of rat liver ferritin. The coding region of 546 nucleotides (182 amino acids) is flanked by 5'- and 3' -untranslated regions of approximately 130 and 150 nucleotides, respectively. The rat liver L-subunit amino acid sequence derived from the reading frame of the cDNA showed 88% and 82% homology, respectively, with the amino acid sequences of horse spleen ferritin (Heusterspreute, M., and Crichton, R. R. (1981) FEBS Lett. 129, 322-327), and human spleen ferritin (Wustefeld, C., and Crichton, R. R. (1982) FEBS Lett. 150, 43-48), thus demonstrating evolutionary conservation of the L-subunit sequence. However, a major difference between the rat and the horse and human sequences is the insertion of an octopeptide near the COOH-terminus of the rat protein resulting in a slightly longer peptide chain in this species. The reading frame and parts of the derived amino acid sequence including the octopeptide sequence were confirmed by direct amino acid sequencing of cyanogen bromide peptides from rat liver ferritin. Minor fragments of rat liver ferritin, presumably derived from the H-subunit, were also isolated after cyanogen bromide treatment. On sequencing, these H-peptides showed limited homology with regions of the L-sequence but extensive homology with published H-sequences from human liver and spleen. The H-subunit sequence did not contain the octopeptide found as part of the L-subunit sequence.

Amino Acid Sequence↗
PubMed1984

Placental lactogen administration reverses the effect of low-protein diet on maternal and fetal serum somatomedin levels in the pregnant rat.

Female rats were studied on day 20 of pregnancy after being fed either a 5% lactalbumin (low protein) diet or a 20% lactalbumin (adequate) diet for the last 2 weeks of pregnancy. Rats on the lower intake of protein showed decreased serum levels of rat placental lactogen and reduced numbers of lactogenic receptors in the maternal liver. These changes were accompanied by much reduced serum levels of somatomedins IGF I(insulin-like growth factor) and II (multiplication-stimulating activity, MSA). Infusion of human placental lactogen or human growth hormone into the rats on the low-protein intake during the last 2 weeks of pregnancy partially restored the maternal serum levels of both somatomedins, but only human placental lactogen increased the number of lactogenic receptors on liver cell membranes. It was concluded that protein deficiency may reduce secretion of somatomedins by the liver (or other tissues) of the pregnant rat indirectly through reduction in output of rat placental lactogen by the placenta. In the same experiments, the effect of maternal protein deficiency on fetal development and serum somatomedin levels was examined. Protein deficiency resulted in smaller fetuses and placentas and lower fetal serum levels of IGF I and MSA. Unlike the response in maternal serum levels, the concentration of MSA in the fetal serum increased during infusion of hPL or hGH but the concentration of IGF I did not. This suggests that placental lactogen enters the fetal circulation and affects tissues producing MSA but not those making IGF I. Despite the restoration of MSA levels, fetal and placental weights did not increase when the rats on the protein-deficient diets were treated with human placental lactogen or growth hormone.

Animals↗