Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor.

Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.

Oxidation of pristanic acid in fibroblasts and its application to the diagnosis of peroxisomal beta-oxidation defects.

Pristanic acid oxidation measurements proved a reliable tool for assessing complementation in fused heterokaryons from patients with peroxisomal biogenesis defects. We, therefore, used this method to determine the complementation groups of patients with isolated defects in peroxisomal beta-oxidation. The rate of oxidation of pristanic acid was reduced in affected cell lines from all of the families with inherited defects in peroxisomal beta-oxidation, thus excluding the possibility of a defective acyl CoA oxidase. Complementation analyses indicated that all of the patients belonged to the same complementation group, which corresponded to cell lines with bifunctional protein defects. Phytanic acid oxidation was reduced in fibroblasts from some, but not all, of the patients. Plasma samples were still available from six of the patients. The ratio of pristanic acid to phytanic acid was elevated in all of these samples, as were the levels of saturated very long chain fatty acids (VLCFA). However, the levels of bile acid intermediates, polyenoic VLCFA, and docosahexaenoic acid were abnormal in only some of the samples. Pristanic acid oxidation measurements were helpful in a prenatal assessment for one of the families where previous experience had shown that cellular VLCFA levels were not consistently elevated in affected individuals.

The effect of clofibrate on the phospholipid composition of the peroxisomal membranes in mouse liver.

Membranes were prepared from peroxisomes which had been isolated from the livers of normal mice and from mice treated with clofibrate (a hypolipidemic drug and peroxisome proliferator). Phospholipid analysis of these membranes revealed that clofibrate treatment resulted in a decrease in the membrane content of phosphatidylcholine, the most abundant phospholipid, and a concomitant increase in the amount of lysophosphatidylcholine, this latter component reaching a level of almost 6% of the total membrane phospholipid. The concentrations of other phospholipids in these membranes were not significantly altered. The parallel analysis of microsomal membranes demonstrated an analogous increase in the level of lysophosphatidylcholine following clofibrate treatment. In control experiments with microsomal membranes employing quinacrine, an inhibitor of phospholipase A2, the increased lysophosphatidylcholine concentration was still observed in clofibrate-treated animals. As well, a decrease in the proportion of microsomal phosphatidylcholine with clofibrate treatment was seen when quinacrine was used. Fatty acid analysis of the phosphatidylcholines from peroxisomal membranes showed some minor changes, including an increase in one component tentatively identified as docosahexaenoic acid, in clofibrate-treated animals. Overall, these data demonstrate that clofibrate causes a marked perturbation of the phospholipid composition of peroxisomal membranes, and are interpreted as indicating that the main site of action of the drug is the deacylation-reacylation cycle between phosphatidylcholine and lysophosphatidylcholine.

Analysis of the major integral membrane proteins of peroxisomes from mouse liver.

Two major proteins with subunit molecular masses of 68 and 70 kDa were isolated from the integral membrane protein fraction of peroxisomes purified from mouse liver. The two proteins were shown to be distinct proteins by two criteria: first, immunoblot analysis demonstrated that antisera against the 68 kDa protein did not cross-react with the 70 kDa protein, and vice versa; and second, the partial peptide maps resulting from proteinase digestion of the proteins were different. Immunoblot analyses to test the specificities of the antisera demonstrated that only the expected molecular mass species in purified peroxisomes, and in membranes prepared from these organelles, were recognized; there was no identification of proteins from purified mitochondrial or microsomal fractions. The concentrations of both of these proteins were increased in livers of mice treated with clofibrate, a hypolipidemic drug and peroxisome proliferator, with the effect being greater for the 70 kDa component. The localization of the 68 kDa protein was shown to be completely integral to the peroxisome membrane. Although some 70 kDa protein was integral to the membrane, a significant proportion was released from the membrane by some procedures believed to detach peripheral proteins. The 70 kDa protein was also particularly susceptible to degradation during isolation - in particular, addition of EDTA to media used for isolation of peroxisomes resulted in membranes in which this protein was degraded to smaller immunoreactive fragments. These data have been discussed in relation to the significant clarification which they have provided of the status and characteristics of the major protein components of peroxisomal membranes.

Identification of a catalase-negative sub-population of peroxisomes induced in mouse liver by clofibrate.

The peroxisomal compartment in mouse liver was investigated using rate sedimentation of liver subfractions on sucrose density gradients. Treatment of mice with clofibrate, a hypolipidemic agent and peroxisome proliferator, resulted in the formation of small particles which were devoid of catalase and urate oxidase, but which were identified as peroxisomal on the basis of content of the clofibrate-induced peroxisomal beta-oxidation enzymes (fatty acyl-CoA oxidase, hydratase/dehydrogenase bifunctional protein, and thiolase) and the 68 kDa peroxisomal integral membrane protein. Immunoelectron microscopy confirmed the membrane-bound organellar nature and enzyme composition of these particles. These particles were absent in normal mice, and were increased to a maximal level within 2 days of clofibrate treatment. These data have been taken as indicative of a role of these particles in the mechanism of drug-induced peroxisome proliferation.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: correlations with fetal growth.

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases ofnoninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-3 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGH at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P < .001), free PGH and total PGH correlated very closely (r = 0.98). The results are consistent with an inhibitory function for GHBP in vivo and support a critical role for placental GH and IGF-I in driving normal fetal growth.

On the role of the peroxisome in ontogeny, ageing and degenerative disease.

This article reviews the available data on the role of the peroxisome in the growth, differentiation and degeneration of mammalian tissues. Developmental progressions of peroxisomes are described, along with the influence of inhibitors of peroxisomal enzymes, peroxisome proliferators and morphogenetic agents on the ontogeny of experimental animals. The role of the peroxisome in protecting tissues from damage by oxygen free radicals is also described, as is the changing role of the peroxisome in the ageing animal. Amongst the degenerative diseases which have been associated with free radical damage are cancer, atherosclerosis, muscular dystrophy, rheumatoid arthritis and the senile degeneration of brain function. In all these conditions, the major characteristics of molecular damage have been considered, along with the particular role of the peroxisome in alleviating these effects. Proposals for further research into peroxisomal function during ontogeny and the degenerative changes associated with ageing are developed, and the possibility of palliative treatments discussed.

Chromatin structure and the expression of cardiac genes.

Actively transcribed genes are more susceptible to nuclease digestion, an observation suggested to reflect an altered state of chromatin organization. It has been hypothesized that exposure or sequestration of chromatin domains is a higher order gene regulatory mechanism. In order to test whether tissue lineage is organized by mechanisms at the level or chromatin structure, three cardiac phenotype-conferring genes (atrial natriuretic factor, myosin light chain-1-ventricular and alpha-tropomyosin) have been assessed for DNase 1 sensitivity in nuclei prepared from tissues of the developing guinea pig. These data have been related to the level of tissue mRNA expression of these genes to ascertain whether the exposed state of genes can occur when transcription is low or undetectable. Although this phenomenon was evident in some cases, the data were not consistent with mechanisms at the level of chromatin structure directing tissue type.

On the ontogeny of cardiac gene transcripts.

As a prerequisite to investigating the specification and differentiation of cardiac tissue in vitro, the ontogeny of a number of putative cardiac-specific, and striated muscle-specific gene transcripts has been studied. The probes used include cDNAs of alpha-actins, myosin heavy chains, myosin light chains, alpha-tropomyosin, troponin-T and atrial natriuretic factor. The expression of these genes was monitored by Northern analysis of heart and various other tissues at three developmental ages, viz, adult, neonatal and mid-foetal. The aim of this exercise was to confirm the efficacy of a number of markers to represent a cardiac-specific subset of gene expression in our mammalian model, the guinea pig. Our results indicate predominantly cardiac expression for the mRNA transcripts of cardiac alpha-actin (c alpha-actin), cardiac myosin heavy chain-alpha (MHC alpha), cardiac myosin heavy chain-beta (MHC beta), myosin light chain-1A (MLC1A), myosin light chain-1V (MLC1V), alpha-tropomyosin (alpha TM), cardiac troponin-T (cTnT) and atrial natriuretic factor (ANF). Furthermore, cardiac-specific expression at the midfoetal time point was observed for five gene transcripts, MLC1V, MHC alpha, MHC beta, striated alpha TM and ANF. No genes were expressed exclusively in cardiac tissue; for example, expression of the genes for c alpha-actin, both cardiac MHCs, both MLCs, alpha TM and cTnT was evident in skeletal and vascular smooth muscles at some stages of development. An interesting difference between this species and those of previous studies was the minor contribution of skeletal alpha-actin to cardiac phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol, alcoholic brain damage, and GABAA receptor isoform gene expression.

Selective variations in cerebral GABAA receptor pharmacology and function are observed in experimental animals subjected to a number of alcohol-treatment and -withdrawal paradigms, and where human alcoholics with and without a range of concomitant diseases are compared with non-alcoholic cases. Recombination studies have shown that variations in GABAA receptor pharmacology and function can result from altering its subunit isoform composition. This commentary examines the rôle of subunit isoform expression in the response to long-term alcohol administration in animals, and in the pathogenesis of alcoholism-related brain damage in human cases.

Expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor in human alcoholic brain.

The expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the alpha class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patient's age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because alpha 1 expression was raised in both groups of alcoholics of controls. The two groups of alcoholics could be differentiated on the basis of regional variations in alpha 1 expression. In frontal cortex, alpha 1 mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, alpha 1 expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform.

Zolpidem binding sites on the GABA(A) receptor in brain from human cirrhotic and non-cirrhotic alcoholics.

The displacement of [3H]flunitrazepam by unlabelled flunitrazepam or zolpidem was used to assess the affinity and density of sub-types of GABA(A) receptors in the superior frontal and primary motor cortices of ten alcoholic, seven alcoholic-cirrhotic and ten matched control cases. The binding was best fitted by a model with a single site for flunitrazepam, but two sites for zolpidem. Neither the patients' age nor the post-mortem interval were significantly correlated with the affinity or density of any of the binding sites. The affinity of all ligands did not differ either between cortical regions or across case groups. Hence, the density of each binding site was analyzed at constant affinity. The densities of flunitrazepam and high-affinity zolpidem binding sites were invariant across cortical regions and case groups. Low-affinity zolpidem binding sites were significantly more dense in the frontal than in the motor cortex of alcoholic cases irrespective of cirrhosis, whereas this regional difference was not significant in control cases.

A method for the quantitation of the alpha 1, alpha 2, and alpha 3 isoforms of the GABAA receptor in human brain using competitive PCR.

The GABAA receptor is the site of action of the inhibitory neurotransmitter GABA, as well as a number of pharmacologically important drugs such as benzodiazepines, barbiturates, and ethanol. The GABAA receptor is a pentameric complex composed of distinct polypeptides, which have been divided into five subunit classes on the basis of sequence homology. To date, 17 isoforms of the receptor have been identified and cloned in mammalian brain, and designated alpha 1-6, beta 1-4, gamma 1-4, delta and rho 1-2. In addition, several isoforms exist in alternatively spliced forms (for review see ref.). Studies on recombinant receptors have revealed that receptors constituted from different isoforms exhibit distinct pharmacological properties. For example, the alpha subunit class appears to be responsible for GABA enhancement of benzodiazepine binding. GABAA receptor function is modulated by benzodiazepine agonists such as flunitrazepam and diazepam, barbiturates, anaesthetics, neurosteroids, and ethanol. Chronic treatment of animals with many of these compounds can bring about profound changes in receptor expression and pharmacology. The RT/PCR assay described here was developed to quantify the alpha 1, alpha 2 and alpha 3 isoforms in the same assay. The amount of each isoform was quantified on the basis of a standard curve generated under identical PCR conditions to the target sequences. In this way it is possible to quantify multiple samples in each RT/PCR assay, thereby reducing inter-assay variability. The assay can be applied to quantify the expression of these isoforms in response to acute and chronic drug administration, or in particular disease states. Altered expression may reflect a corresponding change in protein synthesis, or an alteration of the subtype composition of GABAA receptor.

Isolation and characterisation of a chick cDNA encoding the RNA polymerase common subunit RPB6.

The RPB6 cDNA of chicken, encoding one of the small subunits common to all three nuclear DNA-dependent RNA polymerases, has been isolated from an expression cDNA library by screening with a differential display derived probe, representing a gene shown to be highly up-regulated in early heart development. The nucleotide sequence of the cDNA isolated predicts a protein sequence of 127 amino acids. This sequence shares 124 amino acids (98% homology) with the human RNA polymerase II subunit 14.4 kDa (RPB6) and hamster hRPB6 and 123 amino acids (97% homology) with Rattus norvegicus RNA polymerase II subunit RPB6. Other conserved motifs in this protein and potential functions of RPB6 are discussed.

Characterisation of a cDNA encoding chick eukaryotic translation initiation factor-2 beta.

A full length cDNA for the beta subunit of chick (Gallus gallus) eukaryotic translation initiation factor-2 is described. This cDNA was isolated by screening a chick cDNA library with a probe derived via differential display of developing chick heart tissue. Up-regulated expression of eIF-2 beta mRNA was confirmed by reverse Northern dot blot analysis. eIF-2 beta, together with eIF-2 alpha and eIF-2 gamma, comprise subunits of a complex that promotes the binding of methionyl-tRNA to ribosomes during the initiation of protein translation. The nucleotide sequence of the chick eIF-2 beta cDNA predicts a protein of 334 amino acids that has 95%, 93%, 56% and 37% sequence identity with rabbit, human, drosophila and yeast eIF-2 beta, respectively. The deduced eIF-2 beta protein contains a number of functional motifs and domains consistent with the putative function of this protein; these include a potential C2-C2 zinc-finger binding domain, three polylysine regions, and three acidic regions.

Central serotonergic neuron deficiency in a mouse model of Zellweger syndrome.

Zellweger syndrome (ZS) is a severe peroxisomal disorder caused by mutations in peroxisome biogenesis, or PEX, genes. A central hallmark of ZS is abnormal neuronal migration and neurodegeneration, which manifests as widespread neurological dysfunction. The molecular basis of ZS neuropathology is not well understood. Here we present findings using a mouse model of ZS neuropathology with conditional brain inactivation of the PEX13 gene. We demonstrate that PEX13 brain mutants display changes that reflect an abnormal serotonergic system - decreased levels of tryptophan hydroxylase-2, the rate-limiting enzyme of serotonin (5-hydroxytryptamine, 5-HT) synthesis, dysmorphic 5-HT-positive neurons, abnormal distribution of 5-HT neurons, and dystrophic serotonergic axons. The raphe nuclei region of PEX13 brain mutants also display increased levels of apoptotic cells and reactive, inflammatory gliosis. Given the role of the serotonergic system in brain development and motor control, dysfunction of this system would account in part for the observed neurological changes of PEX13 brain mutants.

Induction of the major integral membrane protein of mouse liver peroxisomes by peroxisome proliferators.

Peroxisome proliferators are known to increase the volume of the peroxisomal compartment in rodent liver. We have examined the induction of the major integral membrane protein of mouse liver peroxisomes (PMP68) by a number of these agents, and compared this with their effect on the peroxisomal bifunctional protein (PBP), an enzyme of the beta-oxidation pathway which is located in the peroxisome matrix. Dietary clofibrate, di-2-(ethylhexyl)phthalate and Wy-14,643, three structurally unrelated proliferators, all increased the mRNA and protein content of PMP68 approx. 2-fold, whereas PBP was induced 8-13-fold. The kinetics and sequence of induction of PMP68 and PBP following a single dose of Wy-14,643 were compared and shown to be similar, and the effects were reversible. Another proliferator, BM 15766, caused maximal induction of PMP68 but only a low induction of PBP; further PBP induction was achieved by the administration of BM 15766 in combination with Wy-14,643. Similarly, BM 15766 and Wy-14,643 increased transcription of the PMP68 gene in vitro, whereas PBP gene transcription was increased by Wy-14,643 but not by BM 15766. Thus peroxisome proliferators enhance the expression of the genes for both the membrane protein PMP68 and the matrix protein PBP, but the regulation of this expression appears to be mediated by different mechanisms.

The Pichia pastoris HIS4 gene: nucleotide sequence, creation of a non-reverting his4 deletion mutant, and development of HIS4-based replicating and integrating plasmids.

We have obtained a clone of the Pichia pastoris HIS4 gene and have determined its nucleotide sequence. Based upon its deduced amino-acid sequence, the product of the P. pastoris HIS4 gene has the same structural organization as the Saccharomyces cerevisiae His4 protein and appears to encode a trifunctional enzyme catalyzing the second (phosphoribosyl-ATP pyrophosphohydrolase), third (phosphoribosyl-AMP cyclohydrolase), and tenth (histidinol dehydrogenase) steps in histidine biosynthesis. The chromosomal copy of the HIS4 gene was disrupted by homologous recombination, creating the strain SGY58. The his4 delta deletion mutation in this strain lacks the entire coding region of this gene and has a reversion rate that is undetectable. A set of complementary plasmids that carry the HIS4 gene was also developed. Among these are nine E. coli-P. pastoris shuttle vectors that transform the his4 delta deletion mutant at high efficiency and an integration vector for creating site-specific alterations of the P. pastoris genome.

Protein organization in mouse liver peroxisomes.

Peroxisomes from mouse liver were fractionated with Triton X-114, a procedure which yields a detergent phase consisting of proteins containing hydrophobic binding sites, and a nondetergent, or aqueous, phase containing hydrophilic proteins. When this method was applied to peroxisomes from control mice, catalase and fatty acyl-CoA oxidase distributed to the aqueous phase, whereas the integral membrane protein, PMP68, and the bifunctional protein were recovered exclusively in the detergent phase. Urate oxidase distributed intermediate between these two phases. With peroxisomes from mice treated with the peroxisome proliferator clofibrate, the bifunctional protein was recovered in both the detergent and the aqueous phases, and urate oxidase was shifted toward the aqueous phase. Other analyses of the subperoxisomal distribution of the bifunctional protein were consistent with a proportion of this protein being tightly associated with the peroxisomal membrane, or with some other uncharacterized, poorly soluble, component. Sucrose gradient centrifugation of the aqueous phase resulting from Triton X-114 fractionation of peroxisomes revealed that a major proportion of catalase, fatty acyl-CoA oxidase, the bifunctional protein, and other unidentified proteins behaved as if associated under these conditions. In this respect, use of a higher concentration of Triton X-114 for peroxisome fractionation led to the partitioning of some catalase and fatty acyl-CoA oxidase to the detergent phase, indicating the presence of some detergent-accessible hydrophobic binding sites even on these proteins. These data have been interpreted as indicating matrix protein associations in vivo, associations which may be responsive to proliferator treatment.