Characterization of the oral and faecal microbiota associated with atopic dermatitis in dogs selected from a purebred Shiba Inu colony.

Atopic dermatitis (AD) is a chronic and relapsing multifactorial inflammatory skin disease that also affects dogs. The oral and gut microbiota are associated with many disorders, including allergy. Few studies have addressed the oral and gut microbiota in dogs, although the skin microbiota has been studied relatively well in these animals. Here, we studied the AD-associated oral and gut microbiota in 16 healthy and 9 AD dogs from a purebred Shiba Inu colony. We found that the diversity of the oral microbiota was significantly different among the dogs, whereas no significant difference was observed in the gut microbiota. Moreover, a differential abundance analysis detected the Family_XIII_AD3011_group (Anaerovoracaceae) in the gut microbiota of AD dogs; however, no bacterial taxa were detected in the oral microbiota. Third, the comparison of the microbial co-occurrence patterns between AD and healthy dogs identified differential networks in which the bacteria in the oral microbiota that were most strongly associated with AD were related to human periodontitis, whereas those in the gut microbiota were related to dysbiosis and gut inflammation. These results suggest that AD can alter the oral and gut microbiota in dogs.

Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-deficient mammalian cell mutant.

Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay. This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites. PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly. A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis. Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.

Identification of three mutant alleles of the gene for mitochondrial acetoacetyl-coenzyme A thiolase. A complete analysis of two generations of a family with 3-ketothiolase deficiency.

3-Ketothiolase deficiency (3KTD) stems from a deficiency of mitochondrial acetoacetyl-coenzyme A thiolase (T2). We analyzed the molecular basis of 3KTD in two generations of a family. A boy (patient 2, GK04), his father (patient 1, GK05), his mother, and his brother were studied; three mutant alleles of T2 gene were identified. Patient 1 is a compound heterozygote: one allele has a point mutation of G to A at position 547 on his T2 cDNA, causing Gly150 to Arg substitution of the mature T2 subunit, and the other allele has GT to TT transition at the 5' splice site of intron 8, causing exon 8's skipping of the T2 cDNA. Patient 2 is also a compound heterozygote: one allele inherited from his mother has AG to CG transition at the 3' splice site of intron 10, causing exon 11's skipping of the T2 cDNA, and the other allele derived from patient 1 has the G to A mutation (Gly to Arg). The brother of patient 2 is an obligatory carrier with the mutant allele causing the exon 8 skipping. This report seems to be the first complete molecular definition of 3KTD at the gene level.

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus.

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p.

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291Asn and 292Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.

Long-term outcome of 6-month maintenance chemotherapy for acute lymphoblastic leukemia in children.

In the treatment of childhood acute lymphoblastic leukemia (ALL), excess shortening of maintenance therapy resulted in high relapse rate, as shown by our previous trial, TCCSG L92-13, in which maintenance therapy was terminated at 1 year from initiation of treatment. In this study, we aimed to confirm the long-term outcome of L92-13, and to identify who can or cannot be cured by shorter duration of maintenance therapy. To obtain sentinel cytogenetics information that had been missed before, we performed genetic analysis with genomic microarray and target intron-capture sequencing from diagnostic bone marrow smear. Disease-free survival (DFS) at 10 years from the end of therapy was 66.0±2.8%. Females (n=138) had better DFS (74.6±3.7%) than males (n=142, 57.5±4.2%, P=0.002). Patients with TCF3-PBX1 (n=11) and ETV6-RUNX1 (n=16) had excellent DFS (90.9±8.7% and 93.8±6.1%, respectively), whereas high hyperdiploidy (n=23) was the most unfavorable subgroup, with 56.6±10.3% of DFS. Short duration of therapy can cure more than half of pediatric ALL, especially females, TCF3-PBX1 and ETV6-RUNX1. Our retrospective observations suggest a gender/karyotype inhomogeneity on the impact of brief therapy.

Comparison of constitutive and inducible levels of expression of peroxisomal beta-oxidation and catalase genes in liver and extrahepatic tissues of rat.

Previous studies from our laboratories have shown that carcinogenic peroxisome proliferators significantly increase the mRNA levels of peroxisomal beta-oxidation genes in the rat liver by enhancing the transcriptional activity. Because of a good correlation between the inducibility of peroxisome proliferation and carcinogenicity of this class of xenobiotics, we proposed that sustained induction of peroxisomal beta-oxidation system and the resultant oxidative stress form the basis for carcinogenesis. Since this concept implies that tumors should develop only in tissues which display maximal peroxisome proliferation, we have now assessed the degree to which catalase and the three beta-oxidation genes are expressed in liver and 12 extrahepatic tissues of adult rats fed for 2 weeks a diet containing 0.025% ciprofibrate (w/w), a peroxisome proliferator. In the ciprofibrate-treated rats, the levels of catalase mRNA increased to less than 2-fold in liver, kidney, intestine, and heart, but no change was detected in other tissues. The mRNA levels of the three genes of beta-oxidation system in the liver of adult rats treated with ciprofibrate increased greater than 20-fold. In contrast, in the kidney, small intestine, and heart the increases in the mRNA levels of all three beta-oxidation genes were small and varied from 2- to 4-fold following ciprofibrate treatment. Ciprofibrate did not significantly increase the levels of these mRNAs in the other nine tissues. These results correlated well with the levels of peroxisomal beta-oxidation activity, peroxisome volume density, and the immunologically quantified proteins in various tissues. These results provide evidence for the presence of beta-oxidation enzymes in peroxisomes of many tissues of rat and for tissue (cell)-specific differences in the inducibility of mRNAs of these beta-oxidation genes. The marked inducibility of beta-oxidation genes in liver and subsequent development of liver tumors support the hypothesis that tumors develop in tissues that show inducibility of peroxisome proliferation vis a vis beta-oxidation system following exposure to peroxisome proliferators.

Occurrence of two 3-hydroxyacyl-CoA dehydrogenases in rat liver.

3-Hydroxyacyl-CoA dehydrogenase was assayed for acetoacetyl pantetheine-reducing and acetoacetyl-CoA reducing activities in rat liver homegenates. Two isoenzymes of the enzyme, types I and II, were distinguished by the following procedures: trypsin treatment, heat treatment, CM-cellulose chromatography, antibody titration, and sucrose density gradient centrifugation of the light mitochondrial fraction. Type I enzyme was localized in mitochondria, and catalyzed the reduction of both acetoacetyl pantetheine and acetoacetyl-CoA. Type II enzyme was found mainly in peroxisomes, accompanied by a low activity in mitochondria or some other organelles, and was active with acetoacetyl-CoA but not with aceto acetylpantetheine. Both isozymes were induced by the administration to the rats of di-(2-ethylhexyl)phthalate, which enhances the peroxisomal beta-oxidation activity, but the extent of the induction of type II enzyme was much higher than that of type I enzyme. The activity of the former was found only in diethylhexylphthalate-treated rats.

Molecular cloning and functional expression of a cDNA for mouse squalene synthase.

Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library. Its nucleotide sequence had an open reading frame fro a 416 amino acid polypeptide (calculated molecular mass, 48 kDa). In vitro transcription of the cDNA followed by in vitro translation produced a protein of the expected size. The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively. Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome. To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of beta-galactosidase. Escherichia coli, which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene.

Characterization of sterol-ester hydrolase in Saccharomyces cerevisiae.

A homgenate of Saccharomyces cerevisiae grown under semi-anaerobic as well as aerobic conditions was found to catalyze the hydrolysis of fatty acid esters of sterols in the presence of Triton X-100. The enzyme levels in cells grown under various conditions were similar and the enzyme had a broad substrate specificity for sterol esters. The enzyme was localized in the mitochondrial fraction for the aerobically grown cells and in the mitochondrial and cytosolic fractions for the semi-anaerobically grown cells.

A novel isoform of rat hepatocyte nuclear factor 4 (HNF-4).

Based on the published nucleotide sequence for rat hepatocyte nuclear factor 4 (HNF-4; Sladek, F.M., Zhong, W., Lai, E. and Darnell, J.E., Jr. (1990) Genes Dev. 4, 2353-2365), we have cloned a cDNA by means of polymerase chain reaction amplification of reverse-transcribed RNA (RT-PCR). Our clone contained an extra segment of 30 bp, which was not found in the previously reported clone, in the coding region near the C-terminus. Further RT-PCR analysis demonstrated that both isoforms of HNF-4 mRNA, i.e., with or without the 30 nucleotide segment, occur in rat liver and kidney, presumably by differential splicing.

cDNA cloning of a putative G protein-coupled receptor from brain.

Using degenerate oligonucleotide primers corresponding to conserved regions of the G-protein coupled receptor superfamily, we carried out a low-stringency polymerase chain reaction and obtained two novel partial-length clones from a rat brain cDNA library. We used one of these clones for conventional library screening and isolated a longer cDNA clone, designated as RBU-15, from another rat brain library. Although RBU-15 was truncated at its 5' end, Northern blot analysis revealed that the gene was expressed in the brain and spleen. Next, we isolated a full-length cDNA clone, designated as HB-954, from a human fetal brain library, using RBU-15 as a probe. The deduced amino acid sequence of HB-954 contained four putative glycosylation sites in the N-terminal part, seven transmembrane domains, and a large cytosolic domain in the C-terminal part. The protein products of RBU-15 and HB-954 likely belong to a distinctive subfamily, because no receptors in the superfamily were found to be closely related to them.

Turnover of enzymes of peroxisomal beta-oxidation in rat liver.

Male Wistar rats were given a diet containing 2% (w/w) di-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal beta-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal beta-oxidation was 5--7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the beta-oxidation system and of catalase decreased to the control levels with a half-life of 2--3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.

Nuclear receptor binding factor-2 (NRBF-2), a possible gene activator protein interacting with nuclear hormone receptors.

A protein named nuclear receptor binding factor-2 (NRBF-2) was identified by yeast two-hybrid screening, as an interaction partner of peroxisome proliferator-activated receptor alpha as well as several other nuclear receptors. NRBF-2 exhibited a gene activation function, when tethered to a heterologous DNA binding domain, in both mammalian cells and yeast.

Identification of two splice isoforms of mRNA for mouse hepatocyte nuclear factor 4 (HNF-4).

Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor involved in the expression of many liver-specific genes. In the preceding communication (Hata, S., Tsukamoto, T. and Osumi, T. (1992) Biochim. Biophys. Acta 1131, 211-213), we reported the presence of two isoforms of mRNA for HNF-4 in rat liver and kidney. The longer isoform contained a segment of 30 bases which was not present in the shorter one. As an initial step to determine whether or not other mammals have these mRNA isoforms, we isolated a cDNA for mouse HNF-4 using the rat HNF-4 gene as a probe. The cDNA had an open reading frame for a 465 amino acid polypeptide. The deduced amino acid sequence was remarkably conserved between mouse HNF-4 and rat HNF-4 (99.6% identical). Moreover, like the cDNA for the larger rat isoform, the mouse cDNA contained an extra segment of 30 bp in the coding region near the C-terminus. Blotting analyses showed that the mRNA is about 3.7 kb in size and that a single copy of the gene is present in the mouse genome. Next we carried out the polymerase chain reaction (PCR) using primers located just upstream and downstream of the extra segment. Two PCR products were amplified from a mouse liver cDNA library. Determination of their nucleotide sequences proved that they exactly corresponded to the two rat isoforms. Finally, we amplified a DNA fragment (1.1 kb in size) from mouse genomic DNA using the same PCR primers as above. Its nucleotide sequence unequivocally confirmed that different splice donor sites were used to generate the two isoforms.

Nucleotide sequence of a cDNA for mouse squalene epoxidase.

A full-length cDNA encoding mouse squalene epoxidase was isolated by screening a mouse liver cDNA library with the rat squalene epoxidase gene as a probe. The cDNA had an open reading frame for a 572 amino acid polypeptide with a calculated molecular mass of 63.8 kDa. The predicted amino acid sequence of the mouse enzyme contained an FAD-binding motif, and was 93% identical to that of the rat enzyme. The former is one amino acid shorter than the latter. Blotting analyses showed that the mRNA is 2.8 kb in size and that a single copy of the gene is present in the mouse genome.

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase.

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.

Nucleotide sequence of the human 70 kDa peroxisomal membrane protein: a member of ATP-binding cassette transporters.

The cDNA sequence of human liver 70 kDa peroxisomal membrane protein (hPMP70) was determined. The nucleotide sequence contains an open reading frame of 1977 base pairs and encodes an amino acid sequence of 659 residues which exhibits 95.0% identity with that of rat liver PMP70. hPMP70 shares close similarity to the members of a superfamily of ATP-binding transport proteins.