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Biomedical subjects

A L Miller

Publications and source records attributed to A L Miller.

At least 145 records · Page 8Linked to original sources
PubMed1984

Identification of mannose 6-phosphate receptors in rabbit alveolar macrophages.

Mannose 6-phosphate is an important recognition site involved in transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes. The current study is the first demonstration of functional mannose phosphate receptors in macrophages. The receptor appears to be similar in many respects to that expressed in fibroblasts. Binding at 4 degrees C of a mannose-6-P-containing ligand, alpha-mannosidase from Dictyostelium discoideum, was specific and saturable (KD = 1.6 nM). In the presence of permeabilizing agents (saponin and digitonin), macrophage mannose-6-P receptors gave a distribution of 15-20% on the surface and 80-85% inside. Uptake studies gave a Kuptake value of 4.9 nM. Mannose-6-P, Hansenula holstii phosphomannan, and fructose 1-phosphate were effective inhibitors of alpha-mannosidase uptake. Inhibitors of mannose uptake, such as beta-glucuronidase, mannose-bovine serum albumin, fucose-bovine serum albumin, or mannan had no effect on alpha-mannosidase uptake. Likewise, an inhibitor (fucoidin) of the macrophage receptor which recognizes negatively charged proteins did not inhibit alpha-mannosidase uptake. Uptake was linear over 90 min and inhibited by chloroquine, suggesting that surface receptors recycle. These data demonstrate that macrophages contain receptors which specifically recognize mannose-6-P units and are distinct from the well characterized mannose receptors. The finding that the mannose-6-P receptors play a role at the surface, together with the fact that most of the receptors are intracellular (similar to the mannose receptor) suggests that both carbohydrate receptors play a regulatory role at the surface and intracellularly in transport of lysosomal enzymes.

Animals↗
PubMed1984

Chronic lymphocytic leukaemia presenting as a blast cell crisis.

A man presented with a short history suggestive of anaemia and thrombocytopenia. Haematological investigations showed him to have an absolute peripheral lymphocytosis and the bone marrow was almost totally replaced by lymphoid blasts. A lymph node biopsy was interpreted as showing immunoblastic lymphoma supervening on chronic lymphocytic leukaemia. Cell surface marker studies performed on cells from peripheral blood and the excised lymph node demonstrated that the lymphocytes and blast cells carried the same surface monoclonal immunoglobulin and expressed OKT1/Leu I positivity. He was treated with a cytotoxic regimen for adult acute lymphoblastic leukaemia but did not respond. This appears to be the first report of a patient with chronic lymphocytic leukaemia who presented at the time of immunoblastic crisis.

Adult↗
PubMed1984

Effects of lactate on glucose metabolism of developing rat brain.

The effects of hyperlactatemia on cerebral glucose metabolism of normoglycemic 20-day-old rats were studied in animals breathing air or 20% CO2:21% O2:59% N2. Sodium lactate or sodium bicarbonate were given intraperitoneally, together with a mixture of [3H]deoxyglucose and [2-14C]glucose. Animals were sacrificed in a freeze-blowing apparatus at intervals of 2-15 min after injection. Blood lactate levels in the lactate-injected rats were 4-6 mM. Hyperlactatemia caused a gradual decline in the brain rate of glucose utilization in air-breathing animals to 50-70% of control rates. Results with both tracers were similar. Concentrations of Krebs cycle intermediates and glutamate did not decrease. These findings indicate that lactate can partially replace glucose as an oxidative fuel for developing rat brain. Hypercapnia depressed the rate of glucose utilization by developing brain and rates were 30-40% lower still in lactate-injected hypercapnic rats. Decreases in levels of Krebs cycle intermediates and glutamate were similar in both groups. Thus, lactate and CO2 are additive in their depressant effects on brain glucose utilization. The observation that lactate did not prevent the decreases in Krebs cycle intermediates and glutamate caused by hypercapnic acidosis suggests an inhibition of flux through pyruvate dehydrogenase during hypercapnia. The data from this study, coupled with data on lactate transport across the blood-brain barrier, indicate that the direction of movement of lactate and its rate of utilization by developing brain are functions of its concentration on blood relative to brain. Physiological and pathological conditions which elevate blood lactate levels above those in brain will, then, have a sparing effect upon brain glucose utilization.

Alkalosis↗
PubMed1984

In vitro expression of suppressogenic and enhancing activities in human colon cancer cells.

The presence of immunoregulatory factors in a human colon cancer cell line, LS174T, was exhibited in mitogenic and immunogenic assays. The simultaneous presence of stimulatory and suppressive factors in the spent media of LS174T colon cancer cells was shown. Opposing suppressive and stimulatory activities were extractable from viable tumor cells and found in 3 M KCl extracts fractionated by isoelectric focusing. Suppressogenic activity was demonstrated in concanavalin A and phytohemagglutinin mitogenesis in both human and murine lymphocytes; the blastogenesis of mixed lymphocyte culture reactions was suppressed, while responses of immune cells were not. Naive lymphocytes were stimulated or not affected by the same spent media. The factors appeared to require cell viability for optimal expression and were not species specific. These results further illustrate the complexity of tumor cell-host cell interactions and emphasize the need for in vitro assays to measure, characterize, and exploit the factors.

Adenocarcinoma↗
PubMed1983

Structural analysis of the asparagine-linked oligosaccharides from three lysosomal enzymes of Dictyostelium discoideum. Evidence for an unusual acid-stable phosphodiester.

Lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-phosphate and bind with high affinity to the phosphomannosyl receptor of human fibroblasts. In this study, we have partially characterized the Asn-linked oligosaccharide units present on these enzymes. [3H]Mannose-labeled alpha-D-mannosidase, beta-D-glucosidase, and beta-D-N-acetylglucosaminidase were purified from the spent growth medium of strain AX3 and glycopeptides were prepared by pronase digestion. Approximately 75% of the glycopeptides contained sulfate residues. These could be removed by solvolysis without degrading the underlying oligosaccharide. Following solvolysis (but not before), the oligosaccharides could be released by endo-beta-N-acetylglucosaminidase H, indicating the presence of high mannose-type units. Greater than 85% of the oligosaccharides contained one or two mannose 6-phosphate residues in the form of an unusual acid-stable phosphodiester. About 3% of the oligosaccharides contained phosphomonoesters and only 6% were neutral species. The major neutral oligosaccharide eluted in the position of Man9GlcNAc when analyzed by high performance liquid chromatography whereas the minor species appeared to be 1-2 residues larger. Acetolysis of the major phosphorylated fractions revealed that molecules with a single mannose 6-phosphate contained the phosphomannosyl residue on the branch linked alpha 1,6 to the beta-linked mannose whereas molecules with two phosphomannosyl residues had the residues on this branch as well as the branch linked alpha 1,3 to the beta-linked mannose. The mechanism of mannose phosphorylation in the slime mold must differ from that of mammalian cells since the phosphomannosyl residues are present as acid-resistant phosphodiesters rather than acid-labile phosphodiesters.

Asparagine↗
PubMed1983

The mod A mutant of Dictyostelium discoideum is missing the alpha 1,3-glucosidase involved in asparagine-linked oligosaccharide processing.

The recessive mutation, mod A, in the Dictyostelium discoideum strain M31 results in an alteration in the post-translational modification of lysosomal enzymes. We now report studies which indicate that mod A is deficient in glucosidase II, an enzyme which is involved in the processing of asparagine-linked oligosaccharides. [2-3H]Mannose-labeled glycopeptides were prepared from three purified mod A lysosomal enzymes and compared to the equivalent glycopeptides from parental enzymes. The mod A glycopeptides were deficient in high mannose oligosaccharides containing two phosphomannosyl residues and accumulated oligosaccharides with one phosphomannosyl residue. The phosphate was present in the form of an acid-stable phosphodiester in both instances. There was also an increase in the amount of nonphosphorylated high mannose oligosaccharides mod A and these were larger than the corresponding material from the parental enzymes. In addition, the nonphosphorylated oligosaccharides were only partially degraded by alpha-mannosidase, indicating the presence of a blocking moiety. In vitro enzyme assays demonstrated that the mod A cells cannot remove the inner 1 leads to 3-linked glucose from a glucosylated high mannose oligosaccharide. The cells are also deficient in membrane-bound neutral p-nitrophenyl-alpha-D-glucosidase activity. This activity has been attributed to glucosidase II in other systems. Removal of the outer 1 leads to 2-linked glucose from Glc3Man9Glc-NAc2 is normal, demonstrating the presence of glucosidase I activity. We conclude from these data that M31 cells are deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the glucosylated oligosaccharides of newly glycosylated proteins. This defect can explain the mod A phenotype and is proposed to be the primary genetic defect in these cells.

Asparagine↗
PubMed1983

Insulin receptor down regulation in human erythrocytes.

Insulin receptor processing in human erythrocytes was investigated. Insulin binding to the cell surface was found to decrease by 70% in cells which had first been incubated with insulin for 3 h, then washed for 3 h. After an additional 16-h incubation without insulin, the level of cell surface insulin binding was restored to control values, even in the presence of cyclohexamide. Our results suggest that erythrocyte insulin receptors are internalized in response to insulin and that receptors are subsequently recycled to the surface of the cell.

Erythrocytes↗
PubMed1983

Incorporation of mannose 6-phosphate receptors into liposomes. Receptor topography and binding of alpha-mannosidase.

A receptor that binds the lysosomal enzyme alpha-mannosidase via mannose 6-phosphate moieties (mannose 6-phosphate receptor) was purified from Swarm-rat chondrosarcoma and bovine liver microsomal membranes. Receptor-reconstituted liposomes were prepared by dialysis of taurodeoxycholate-dispersed lipids with purified mannose 6-phosphate receptor. Liposomes appeared by electron microscopy as 60-120 nm unilamellar vesicles. Receptor-reconstituted liposomes retained the ability to bind alpha-mannosidase specifically. Binding was saturable with an apparent Kd of 1 nM and was competitively inhibited by mannose 6-phosphate (Ki 2mM). Liposomes containing entrapped 125I-bovine serum albumin were used to demonstrate that treatment with 0.045% taurodeoxycholate rendered liposomes permeable to macromolecules without solubilizing the membrane. Receptor orientation in the liposome membrane was established by measuring binding of ligand to intact and detergent-treated liposomes. Unlike coated vesicles, which contain cryptic mannose 6-phosphate receptors [Campbell, Fine, Squicciarini & Rome (1983) J. Biol. Chem. 258, 2526-2533], treatment of liposomes with detergent revealed no additional cryptic binding sites. In addition, treatment of liposomes with 0.75% trypsin abolished total receptor binding activity. The results suggest that the receptor is inserted with its binding site facing the outside of the liposome.

Animals↗
PubMed1983

Uptake of alpha-D-mannosidase and beta-D-glucosidase from Dictyostelium discoideum via the phosphohexosyl receptor on normal human fibroblasts.

alpha-D-Mannosidase and beta-D-glucosidase from Dictyostelium discoideum are efficiently endocytosed into mutant human fibroblasts through a saturable, mannose 6-phosphate (Man-6-P)-inhibitable uptake system (Freeze, H. H., Kaplan, A., and Miller, A. L. (1980) J. Biol. Chem. 255, 11081-11084). We have extended this study using both of these active, purified enzymes and 125I-labeled beta-glucosidase for uptake into normal human fibroblasts. The pH optimum of uptake is 6.0 for both enzymes and greater than 95% is inhibited by 2 mM Man-6-P (Ki = 5 X 10(-5) M). A variety of mono-and diesterified mannans or mannan derivatives also inhibited uptake of the enzymes. Both enzymes compete with each other for uptake (Ki, 2.0 X 10(-9) M) and have Kuptake of 1.0-2.2 X 10(-9) M and a Vmax of 0.35-0.48 pmol/mg of cell protein/h. The specific binding of 125I-beta-glucosidase to fibroblasts was measured at 0-4 degrees C and found to have a Kd of 1.0 X 10(-9) M with approximately 15,900 +/- 900 receptors/cell. The receptors could be internalized every 5-7 min at saturating concentrations of enzyme at 37 degrees C. The 125I-beta-glucosidase previously bound to the cells at 4 degrees C could be released by continued incubation at 4 degrees C in the presence of Man-6-P, however, after brief warming to 37 degrees C followed by reincubation at 4 degrees C, Man-6-P could no longer release the ligand. Chloroquine inhibited 95% of the uptake of 125I-beta-glucosidase at 50 microM. Following internalization of the enzyme, it is degraded to trichloroacetic acid-soluble fragments with a half-life of approximately 6.5 h. These data suggest that the slime mold enzymes are bound to the same receptors which function in the uptake of mammalian lysosomal enzymes and make the slime mold lysosomal enzymes useful models to study uptake involving this receptor in normal human fibroblasts.

Biological Transport↗
PubMed1983

A simple numerical discriminant in the follow-up of lung cancer.

Statistical analysis of laboratory data obtained during a study on a group of patients with lung cancer suggests that a simple numerical discriminant may have value in the follow-up of such patients. The discriminant is based on six common laboratory tests and is rapidly derived in the consulting room using a simple pocket calculator.

Follow-Up Studies↗
PubMed1983

Mucolipidosis II and III. The genetic relationships between two disorders of lysosomal enzyme biosynthesis.

The genetic relationships between the multiple variants of mucolipidosis II (I-cell disease) and mucolipidosis III (pseudo-Hurler polydystrophy) were investigated with a sensitive genetic complementation analysis procedure. These clinically distinct disorders have defects in the synthesis of a recognition marker necessary for the intracellular transport of acid hydrolases into lysosomes. Both disorders are associated with an inherited deficiency of a uridine diphosphate-N-acetyl-glucosamine: lysosomal enzyme precursor N-acetyl-glucosamine-phosphate transferase activity. We had previously shown that both disorders are genetically heterogeneous. Complementation analysis between mucolipidosis II and III fibroblasts indicated an identity of mucolipidosis II with one of the three mucolipidosis III complementation groups (ML IIIA), suggesting a close genetic relationship between these groups. The presence of several instances of complementation within this group suggested an intragenic complementation mechanism. Genetic complementation in heterokaryons resulted in increases in N-acetyl-glucosamine-phosphate transferase activity, as well as in the correction of lysosomal enzyme transport. This resulted in increases in the intracellular levels of several lysosomal enzymes and in the correction of the abnormal electrophoretic mobility pattern of intracellular beta-hexosaminidase. The findings demonstrate that a high degree of genetic heterogeneity exists within these disorders. N-acetyl-glucosamine-phosphate transferase is apparently a multicomponent enzyme with a key role in the biosynthesis and targeting of lysosomal enzymes.

Cell Fusion↗
PubMed1983

Cellular location of N-acetyltransfer activities toward glucosamine and glucosamine-6-phosphate in cultured human skin fibroblasts.

The intracellular location in normal human cultured skin fibroblasts of the N-acetyltransferase activities that transfer the acetyl group from acetyl-CoA to the 2-amino group of glucosamine and glucosamine-6-phosphate have been investigated. Organelles have been separated using a combination of differential centrifugation and free flow electrophoresis. The intracellular distribution of the enzyme involved in the N-acetyltransfer to glucosamine and an alpha-glucosaminide disaccharide indicated that this enzyme activity concentrates mainly with lysosomal organelles whereas the activity associated with N-acetyltransferase to glucosamine-6-phosphate is non-lysosomal. It is proposed that acetyl-CoA: alpha-glucosaminide N-acetyltransferase may be used as a convenient enzyme marker of lysosomal organelle membranes.

Acetyltransferases↗
PubMed1982

Mucolipidosis III beta-N-acetyl-D-hexosaminidase A. Purification and properties.

Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to one of these secreted enzymes, beta-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III urinary beta-N-acetyl-d-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta- d-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000-58000, 55000 and 29000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary beta-N-acetyl-d-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III beta-N-acetyl-d-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-beta-N-acetyl-d-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III beta-N-acetyl-d-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. Our results indicate that as a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-molecular-weight subunits from the cell.

Carbohydrates↗
PubMed1982

A study of highly purified mucolipidosis III urinary N-acetyl-beta-D-hexosaminidase B.

Highly purified N-acetyl-beta-D-hexosaminidase B from normal urine and urine of a patient with mucolipidosis III was used to determine whether it has undergone any of the alterations associated with this genetic defect. Examination by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that both the enzyme preparations contained protein components with apparent Mr values of 55 000 and 28 000. No differences in the binding and apparent KI (50%) to concanavalin A of the normal and mucolipidosis III enzymes were detected. However, the patient's N-acetyl-beta-D-hexosaminidase B had a slightly greater affinity for the lectin from Ricinus communis than did the normal enzyme. Two-dimensional tryptic peptide maps of the corresponding normal and the patient's N-acetyl-beta-D-hexosaminidase B subunits showed considerable homology. These results indicate that N-acetyl-beta-D-hexosaminidase b does not undergo the significant carbohydrate alterations characteristic of other acid hydrolases in mucolipidosis III.

Concanavalin A↗
PubMed1982

Defective catabolism of low-density lipoprotein by fibroblasts from patients with I-cell disease.

Skin fibroblast cultures from patients with I-cell disease (mucolipidosis II) are characterized by multiple lysosomal enzyme deficiencies The present studies deal with the consequences of these deficiencies with respect to the metabolism of plasma low-density lipoproteins. Degradation of the protein moiety was defective in I-cells compared with control cells, but the binding and internalization of low density lipoprotein were much less affected. Measurements of low-density lipoprotein degradation in homogenates demonstrated directly for the first time a deficiency of acid proteinase activity in I-cell fibroblasts. Comparison of results in 6-h incubations with those in 24-h incubations showed accumulation of intracellular low-density lipoprotein in I-cell fibroblasts and an accelerating rate of degradation, possibly attributable to intracellular accumulation of low-density lipoprotein substrate. The significance of these findings with respect to low-density lipoprotein metabolism in vivo is discussed.

Cells, Cultured↗
PubMed1982

Genetic heterogeneity of I-cell disease is demonstrated by complementation of lysosomal enzyme processing mutants.

I-cell disease (mucolipidosis II) is a fatal childhood disorder affecting the expression of multiple lysosomal acid hydrolases. The disorder is characterized by clinical and biochemical heterogeneity which may reflect different mutants with a similar phenotype. Genetic complementation studies demonstrating genetic heterogeneity within this disorder are described utilizing cultured fibroblasts from 11 different patients. Fibroblasts from I-cell disease (ICD) and from five different lysosomal storage diseases with single structural gene enzyme deficiencies were fused in different combinations, and fractions enriched for multinucleated heterokaryons were isolated and tested for acid hydrolase activity and electrophoretic mobility. In fusions of ICD fibroblasts and various single lysosomal enzyme-deficient fibroblasts, the activity of the deficient enzyme and of the other ICD hydrolases were restored, demonstrating that ICD is not a lysosomal enzyme structural gene defect and that the ICD defect, and not just the single enzyme deficiency, is corrected. In fusions involving only I-cell fibroblasts, at least two complementation groups were identified by the recovery of activities of all lysosomal enzymes tested in heterokaryons. These results demonstrate the existence of genetic heterogeneity within the disorder and suggest that different mutations can result in the I-cell clinical and biochemical phenotype. The data support an altered post-translational processing of lysosomal enzymes as the cause of ICD and suggest that at least two genes participate in this pathway.

Cell Fusion↗
PubMed1982

Mucolipidosis III is genetically heterogeneous.

Mucolipidosis III (ML III), or pseudo-Hurler polydystrophy, is an inherited childhood disorder characterized biochemically by low activities and abnormal electrophoretic patterns of multiple lysosomal enzymes in fibroblasts. The primary deficiency of ML III has been proposed to be in UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. However, variation in this enzyme and in other biochemical properties of different ML III lines has been observed. Therefore, we investigated genetic heterogeneity within the disorder by complementation analysis. Heterokaryon cell fractions were generated by fusing together ML III fibroblast lines. When pairs of cells complemented, correction of lysosomal enzyme activities and electrophoretic patterns was observed. Twelve fibroblast lines from 10 sibships were analyzed and three distinct complementation groups were characterized. One complementation group represents the classical ML III disorder. A single cell line identifies a second complementation group. The cell lines comprising a third complementation group have a number of biochemical characteristics different from classical ML III and may represent a genetically distinct disorder.

Cell Line↗