Bovine mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis IIIB, an autosomal recessive lysosomal storage disorder of heparan sulfate caused by mutations in the alpha-N-acetylglucosaminidase (NAGLU) gene, was recently discovered in cattle. Clinical signs include progressive ataxia, stumbling gait, swaying and difficulty in balance and walking. These clinical signs are usually first observed at approximately 2 years of age and then develop progressively over the lifespan of the animals. Affected bulls were found to be homozygous for the missense mutation E452K (c.1354G > A). The availability of mutational analysis permits screening for the NAGLU mutation to eradicate this mutation from the cattle breeding population.

The effect of obesity on the ratio of type 3 17beta-hydroxysteroid dehydrogenase mRNA to cytochrome P450 aromatase mRNA in subcutaneous abdominal and intra-abdominal adipose tissue of women.

OBJECTIVES: To investigate (1) whether type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), the enzyme which catalyzes the conversion of androstenedione to testosterone in the testis, is co-expressed with P450aromatase in the preadipocytes of women, and (2) whether the relative expression of type 3 17beta-HSD and aromatase varies in subcutaneous abdominal vs intra-abdominal adipose tissue of women. SUBJECTS: Subcutaneous abdominal and intra-abdominal adipose tissue was obtained from women undergoing elective abdominal surgery (age 22-78 y, body mass index (BMI) 22.4-52.9 kg/m(2)). MEASUREMENTS: Expression of type 3 17beta-HSD in adipose cell fractions was determined using RT-PCR. Preadipocyte steroidogenesis was investigated in primary cultures using androstenedione as substrate. Messenger RNA levels for type 3 17beta-HSD and aromatase were measured in adipose tissue from the subcutaneous abdominal and intra-abdominal depots using a quantitative multiplex competitive RT-PCR assay. RESULTS: Type 3 17beta-HSD is co-expressed with aromatase in the abdominal preadipocytes of women. Cultured preadipocytes from both subcutaneous abdominal (n=5) and intra-abdominal (n=5) sites converted androstenedione to testosterone, and there was minimal conversion of androstenedione to estrone. Consistent with this, the levels of type 3 17beta-HSD mRNA were significantly higher than aromatase mRNA at both sites (P<0.05; n=8 subcutaneous abdominal, n=12 intra-abdominal adipose tissue). The ratio of levels of 17beta-HSD mRNA to aromatase mRNA in intra-abdominal adipose tissue was positively correlated with BMI (n=11, r=0.61, P<0.05) and waist circumference (n=10, r=0.65, P<0.05). The converse was found in subcutaneous abdominal adipose tissue. CONCLUSION: The intra-abdominal adipose tissue of women may be substantially androgenic, increasingly so with increasing obesity, particularly central obesity. While androgen production by this adipose tissue deposit may not contribute to circulating testosterone levels due to hepatic clearance, it may have hitherto unrecognised local effects in the intra-abdominal adipose tissue and also on the liver via the hepatic portal system. These studies suggest a mechanism linking central obesity with insulin resistance and dyslipidaemia.

Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5'[gamma-thio]triphosphate.

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.

Injection of rat hepatocyte poly(A)+ RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels.

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP3F) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin- and InsP3F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less thari 5 microM Gd3+ for InsP3F-stimulated Ca2+ inflow). GTP gamma S-stimulated Ca2+ inflow was insensitive to 50 microM Gd3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca(2+)-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)+ RNA exhibited greater rates of Ca2+ and Mn2+ inflow in the basal (no agonist) state, greater rates of Ca2+ inflow in the presence of vasopressin or InsP3F and greater rates of Ba2+ inflow in the presence of InsP3F, when compared with "mock"-injected oocytes. In poly(A)+ RNA-injected oocytes, vasopressin- and InsP3F-stimulated Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.

X-linked pyridoxine-responsive sideroblastic anemia due to a Thr388-to-Ser substitution in erythroid 5-aminolevulinate synthase.

BACKGROUND: X-linked sideroblastic anemia is usually associated with reduced 5-aminolevulinate synthase activity in erythroid cells, and some cases are responsive to treatment with pyridoxine, the precursor to the cofactor of the enzyme. The recently identified gene for an erythroid-specific 5-aminolevulinate synthase isoenzyme and its localization to the X chromosome make it likely that one or more defects in this gene underlie the anemia. METHODS: Using a polymorphic dinucleotide-repeat sequence in the erythroid 5-aminolevulinate synthase gene, we confirmed the linkage of this gene to the disorder in a family with X-linked pyridoxine-responsive sideroblastic anemia. We therefore sought evidence of a nucleotide-sequence abnormality in the erythroid 5-aminolevulinate synthase gene by analyzing enzymatically amplified DNA. RESULTS: DNA-sequencing studies in two affected males and one carrier female in the kindred demonstrated a cytosine-to-guanine change at nucleotide 1215 (in exon 8). This change results in the substitution of serine for threonine at amino acid residue 388, near the lysine that binds the pyridoxal phosphate cofactor. In expression studies, the activity of the mutant enzyme was reduced relative to that of the wild type, and this reduction was comparable to that in erythroid cells of the proband during relapse of the anemia; the enzyme activity expressed in the presence of pyridoxine was comparable to that in the proband's marrow cells during remission. Although the affinity of the mutant enzyme for pyridoxal phosphate was not altered, the mutation appears to introduce a conformational change at the active site of the enzyme. CONCLUSIONS: We identified a point mutation resulting in an amino acid change near the pyridoxal phosphate-binding site of the erythroid 5-aminolevulinate synthase isoenzyme as the underlying defect in a kindred with X-linked pyridoxine-responsive sideroblastic anemia.

Human erythroid 5-aminolevulinate synthase. Gene structure and species-specific differences in alternative RNA splicing.

Erythroid 5-aminolevulinate synthase (ALAS) is expressed exclusively in differentiating erythroid cells as the principal isoform of the enzyme to catalyze the first step of the heme biosynthetic pathway. The human gene encoding this isozyme was isolated from a cosmid library, and its structure was characterized with restriction mapping followed by sequencing of fragments. The gene is 22 kilobases long and has 11 exons. Exon 2 encodes the N-terminal signal sequence required for mitochondrial import, exons 3 and 4 encode a variable portion of the N-terminal end, and exons 5-11 the highly conserved C-terminal portion of the mature protein, respectively. Enzymatic amplification of human reticulocyte RNA using PCR techniques revealed two erythroid ALAS mRNA transcripts predicted to encode both the prototypical 64-kDa isoform as well as a novel smaller isoform with a deletion of 37 amino acids near the N terminus. The two mRNA isoforms are generated by alternative splicing of exon 4 and are expressed in fetal erythroid cells as well as at all stages of erythroid development tested, so that there is no evidence of differentiation-specific regulation of exon 4 splicing. However, striking species-specific differences were observed in that alternative splicing of exon 4 was found in man but not dog or mouse; also, the previously described alternative splicing within exon 3 in mouse was not observed in man. This transcript heterogeneity suggests the existence of erythroid ALAS protein isoforms with potentially distinct functional or regulatory roles. The occurrence of species-specific splicing in the least conserved region of the enzyme may reflect another mechanism of gene evolution in eukaryotes.

Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA.

5-Aminolevulinate synthase (ALAS) catalyzes the first step of the heme biosynthetic pathway. cDNA clones for the human erythroid ALAS isozyme were isolated from a fetal liver library. It can be deduced that the erythroid ALAS precursor protein has a molecular weight of 64.6 kd, and is similar in size to the previously isolated human housekeeping ALAS precursor of molecular weight 70.6 kd. The mature mitochondrial forms of the erythroid and housekeeping ALAS isozymes are predicted to have molecular weights of 59.5 kd and 64.6 kd, respectively. The two isozymes show little amino acid identity in their N-terminal signal sequences but have considerable sequence identity in the C-terminal two-thirds of their proteins. An analysis of the immediate promoter of the human erythroid ALAS gene revealed several putative erythroid-specific cis-acting elements including both a GATA-1 and an NF-E2 binding site. An iron-responsive element (IRE) motif has been identified in the 5'-untranslated region of the human erythroid ALAS mRNA, but is not present in the housekeeping ALAS mRNA. Gel retardation experiments established that this IRE motif formed a protein - RNA complex with cytosolic extracts from human K562 cells and this binding was strongly competed with IRE transcripts from ferritin or transferrin receptor mRNAs. A transcript of the ALAS IRE, mutated in the conserved loop of the IRE, did not readily form this protein - RNA complex. These results suggest that the IRE motif in the ALAS mRNA is functional and imply that translation of the mRNA is controlled by cellular iron availability during erythropoiesis.

X-linked sideroblastic anemia and ataxia: linkage to phosphoglycerate kinase at Xq13.

Molecular linkage analysis was performed on a kindred with X-linked sideroblastic anemia and ataxia. Two-point analysis with a DNA probe for phosphoglycerate kinase (PGK1), which maps to Xq13, suggested linkage to the disorder by a lod score of at least 2.60 at a recombination fraction of zero. The disease in this kindred appears to be clinically and genetically distinct from that in previously reported families with X-linked hereditary ataxia or spastic paraparesis. No mapping data are available for inherited X-linked sideroblastic anemia without neurologic abnormalities. However, structural alterations of band Xq13 may be involved in the development of idiopathic acquired sideroblastic anemia. No alterations in the restriction patterns of two X-linked genes involved in erythrocyte formation-i.e., a DNA-binding protein (GF-1) and 5-aminolevulinate synthase (ALAS)-were detected in DNA from affected males, arguing against a large deletion in either of these candidate genes.

Hemin administration to rats reduces levels of hepatic mRNAs for phenobarbitone-inducible enzymes.

The levels of hepatic mRNAs for several enzymes involved in drug metabolism were measured following administration to rats of either phenobarbitone or 2-allyl-2-isopropylacetamide. There was a substantial elevation in the mRNA levels for cytochromes P450 IIB1, IIB2, and IIIA1, epoxide hydrolase, glutathione-S-transferase Ya/Yc subunit, UDP-glucuronosyltransferase isoenzyme (UDPGTr-2), NADPH-cytochrome P450 oxidoreductase, and 5-aminolevulinate synthase. When rats were treated with hemin, together with inducing drug, there was a marked reduction in the induced levels of these mRNAs, with decreases in the range of 55-95%. Basal levels of these mRNAs in the noninduced rat liver were also lowered by hemin administration. Nuclear run-on transcriptional experiments showed that hemin administration substantially lowered both the basal and drug-induced transcriptional activities of the genes for cytochrome P450IIB1/IIB2 and 5-aminolevulinate synthase. In contrast, the mRNA for heme oxygenase was elevated by hemin treatment, whereas the mRNA levels of beta-actin, albumin, and ornithine transcarbamylase, used as controls, were not affected. Treatment of rats with clofibrate resulted in increased levels of mRNA for cytochrome IVA1 and, in addition, those for cytochromes P450IIB1 and P450IIB2. Hemin administration repressed the induction of mRNA levels for cytochromes P450IIB1 and IIB2 but not that for cytochrome P450 IVA1. Additionally, the induction of P450IAI by beta-naphthoflavone was not affected by hemin. The results suggest that heme may negatively control the induction of cytochromes P450IIB1 and IIB2 and other hepatic enzymes by phenobarbitone and phenobarbitone-like drugs and perhaps play a role in regulating drug metabolism. There is, however, no evidence at present as to whether heme has a direct role in such a mechanism or whether injected hemin promotes a secondary response.

Molecular regulation of 5-aminolevulinate synthase. Diseases related to heme biosynthesis.

All nucleated animal cells synthesize heme to provide the prosthetic group of respiratory cytochromes. Large amounts of heme are synthesized by erythroid cells for hemoglobin production and by liver cells for drug-induced cytochromes P450. This review focuses on the first enzyme of the heme biosynthetic pathway, 5-aminolevulinate synthase (ALAS), which catalyzes the rate-controlling step in liver and possibly other tissues. We report that there are two distinct human genes for ALAS: one, a housekeeping gene, is probably ubiquitously expressed while the other is active only in erythroid tissue. By contrast it has been reported that, for porphobilinogen deaminase, the third enzyme of the heme pathway, there is a single human gene with two promoters; one functional in all tissues, the other erythroid specific. In liver, transcription of the housekeeping ALAS gene is induced by drugs and repressed by heme. Heme also acts in a novel way to prevent transport of ALAS into mitochondria, its site of function. Porphyrias result from inherited defects in enzymes of the heme pathway subsequent to ALAS and the molecular abnormality is now known for the most common subtype of acute intermittent porphyria. In developing red cells, levels of ALAS are regulated by increased gene transcription and by a post-transcriptional mechanism, in which iron most probably controls translation of erythroid ALAS mRNA through an iron-responsive element identified in the 5' untranslated region of the mRNA. The human erythroid ALAS gene is located on the X-chromosome, suggesting that a defect in this gene may be responsible for X-linked sideroblastic anemias.

Erythroid 5-aminolevulinate synthase is located on the X chromosome.

The gene for erythroid 5-aminolevulinate synthase has been mapped to Xpter-Xq26 by Southern blot hybridization analysis of a mouse/human hybrid cell panel. In situ hybridization maps the gene to Xp21-Xq21, with the most likely location being on band Xp11.2. The mapping of the erythroid 5-amino-levulinate synthase gene to the X chromosome suggests that a defect in this gene may be the primary cause of X-linked sideroblastic anemia.

Drug induction of P450IIB1/IIB2 and 5-aminolevulinate synthase mRNAs in rat tissues.

Expression of the phenobarbitone-inducible cytochrome P-450 mRNA species (cytochrome P450IIB1/IIB2) has been investigated in tissues of rats following administration of 2-allyl-2-isopropylacetamide or phenobarbitone. Using a cDNA probe complementary to these mRNAs, a 2.1 kb mRNA species was detected in liver and lung of untreated rats and to a lesser extent in kidney. This species was not detected in testis, brain or erythroid spleen. Following drug treatment, this mRNA species was increased in liver and kidney, particularly after administration of 2-allyl-2-isopropylacetamide. Using oligomeric probes specific for cytochrome P-450 IIB1/IIB2 mRNAs it was shown that both mRNAs are present in untreated liver and are induced by drug treatment, while in lung only cytochrome P-450 IIB1 was detected and this was not induced. However, in kidney, neither of these mRNAs could be detected, even after drug treatment. This indicates that either the levels of these mRNAs in kidney are below the limit of detection or that a related mRNA is induced which does not hybridize to the oligomeric probes. This represents the first report of a phenobarbitone-inducible cytochrome P-450 mRNA in rat kidney. The tissue-specific drug induction of cytochrome P-450 mRNAs correlated with an increased level of mRNA for 5-aminolevulinate synthase. This finding is compatible with the proposal that induction of 5-aminolevulinate synthase is dependent upon the lowering of heme levels through increased amounts of cytochrome P-450 apoprotein.

Heme may not be a positive regulator of cytochrome-P450 gene expression.

It has been proposed that transcription of cytochrome-P450 genes is positively regulated by heme, the prosthetic group of cytochrome-P450 proteins. We have investigated this proposal in rats treated with succinylacetone, a known specific inhibitor of the heme biosynthetic pathway. While 2-allyl-2-isopropylacetamide, phenobarbitone, dexamethasone, beta-naphthoflavone and clofibrate induced specific cytochrome-P450-mRNA species in rat liver, the levels of these induced mRNAs were not affected by succinylacetone administration. Synthesis of the first enzyme of the heme biosynthetic pathway, 5-aminolevulinate synthase, is known to be regulated by the end-product heme, with heme inhibiting 5-aminolevulinate-synthase-gene transcription. Hepatic 5-aminolevulinate-synthase mRNA was induced by drugs and the level increased further by succinylacetone. Furthermore, treatment of rats with succinylacetone alone resulted in elevated levels of 5-aminolevulinate-synthase mRNA but did not affect cytochrome-P450-mRNA levels. The results show that while lowered heme levels lead to an increase in 5-aminolevulinate-synthase-mRNA synthesis there is no effect on cytochrome-P450-mRNA levels, implying that heme is not required for the cytochrome-P450-gene transcription.

Regulation of genes associated with drug metabolism.

Exposure of animals to foreign chemicals results in the induction of many enzymes. The mitochondrial enzyme 5-aminolaevulinate synthase (ALV-S) is induced to supply haem for cytochrome P-450 (P-450) enzymes, the key proteins in drug detoxification. The drugs phenobarbital and 2-allyl-2-isopropylacetamide (AIA), although structurally different, elevate levels of ALV-S and a specific class of P-450s, the phenobarbital-inducible P-450s. Synthesis of ALV-S is negatively controlled by the end-product haem and it is proposed that drugs induce P-450 apoprotein which sequesters haem. Studies at the mRNA level show that ALV-S and P-450 are drug induced in a highly tissue-specific fashion and that haem represses mRNA levels in all but erythroid tissues. In liver, drugs activate ALV-S gene transcription and haem represses, but this mechanism does not operate in erythroid cells. Studies on the drug-induction mechanism of phenobarbital-inducible P-450s in chick embryo liver show that increased mRNA levels result from P-450 gene activation together with a significant post-transcriptional mechanism.

5-Aminolevulinate synthase is at 3p21 and thus not the primary defect in X-linked sideroblastic anemia.

The gene for 5-aminolevulinate synthase (ALAS) has been mapped to 3pter-3q13.2 by Southern blot hybridization analysis of a mouse/human hybrid cell panel. In situ hybridization maps the gene to 3p21, distal to the common fragile site at 3p14.2 (FRA3B). The mapping of this gene to an autosome makes it improbable that it is the site of the primary defect in X-linked sideroblastic anemia.

Regulation of 5-aminolevulinate synthase mRNA in different rat tissues.

cDNA clones for rat liver 5-aminolevulinate synthase have been isolated and used to examine mRNA levels in different rat tissues. Northern hybridization analysis of total RNA from various rat tissues showed the presence of a single 5-aminolevulinate synthase mRNA species of estimated length 2.3 kilobases. Primer extension and RNase mapping studies indicated that the mRNA is identical in all tissues. Highest basal levels were seen in liver and heart. Administration of hemin to rats reduced the basal level of this mRNA only in liver but the heme precursor, 5-aminolevulinate (or its methyl ester), repressed the basal levels in liver, kidney, heart, testis, and brain. The drug 2-allyl-2-isopropylacetamide increased the mRNA level in liver and kidney only while human chorionic gonadotropin hormone elevated the level in testis. Administration of the heme precursor 5-aminolevulinate prevented these inductions. Nuclear transcriptional run-off experiments in liver cell nuclei showed that 2-allyl-2-isopropylacetamide and 5-aminolevulinate exert their effect by altering the rate of transcription of the 5-aminolevulinate synthase gene. The results indicate that a single 5-aminolevulinate synthase mRNA is expressed in all tissues and that its transcription is negatively regulated by heme.