We cloned a cDNA coding for a putative human heme oxygenase isozyme, designated type 2 (HO-2), and analyzed its function by transient expression assays. HeLa cells transfected with either HO-2 cDNAs or a cDNA coding for authentic heme oxygenase (HO-1) expressed the activity of heme oxygenase, although no activity was detected in the mock transfected cells. Using specific anti-HO-1 antibody, we showed that expression of a HO-1 cDNA resulted in the increase in its protein levels, but HO-1 protein was not detectable in the cells expressing HO-2 cDNAs. We thus confirmed the functional identity of HO-1 and HO-2. Then, we analyzed their expression in an erythroid cell line, YN-1-0-A. Treatment with hemin or by heat shock (42 degrees C) led to a remarkable increase in the HO-1 mRNA levels, while HO-2 mRNA expression was not induced at all, suggesting that they are under separate regulation.

Summary. The retinoblastoma protein-interacting zinc finger gene (RIZ), a member of the nuclear protein methyltransferase superfamily, is characterized by the presence of the N-terminal PR domain. The RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the PR (PRDI-BF1 and RIZ homologous) domain, RIZ2 lacks it. RIZ gene expression is altered in a variety of human cancers and RIZ1 is now considered to be a candidate tumour suppressor. To investigate the role of RIZ in leukaemogenesis, we analysed the differential expression of RIZ1 and RIZ2 by quantitative real-time reverse-transcription polymerase chain reaction assay. Our results showed that the expression of RIZ1 was significantly decreased in leukaemia cell lines (14 out of 17, 82%) and in patients with acute myeloblastic leukaemia (eight out of 14, 57%). In contrast, RIZ2 expression was increased in patients with acute lymphoblastic leukaemia (eight out of 11, 73%), compared with normal bone marrow cells. These findings indicate that suppression of RIZ1 expression or enhancement of RIZ2 expression may have an important role in leukaemogenesis.

The properties of 9 ␦-aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and purified by glutathioneaffinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and delTC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions in the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified in ADP and indicates the highly heterogeneous nature of mutations in this disorder. IntroductionDelta-aminolevulinate dehydratase (ALAD) deficiency porphyria (ADP) is an autosomal recessive disorder caused by a homozygous ALAD deficiency. Patients with this condition have clinical symptoms of acute hepatic porphyria such as abdominal pain, vomiting, pain in the arms and legs, and neuropathy. ALAD is the second enzyme in the heme biosynthetic pathway. It catalyzes the Knorr-type condensation of 2 molecules of aminolevulinate acid (ALA) to form a monopyrrole, porphobilinogen (PBG). 1 This enzyme activity is present in large excess compared to other enzymes in the heme biosynthetic pathway, and its partial deficiency does not usually result in any clinical consequences. Seven patients with ADP have been reported to date, 2-6 though only 4 of them have been confirmed by immunochemical or molecular analysis. [2][3][4] Of these 4 cases, the first 2 were German patients reported by Doss and coworkers, 7 the third was a Swedish baby boy studied by Thunell and colleagues, 8 and the fourth was an elderly Belgian man reported by Hassoun and associates. 9 Recently, an asymptomatic Swedish baby girl has been identified to have a markedly decreased ALAD activity. 10 Thus far, a total of 9 point mutations of the alad gene have been identified in these 5 subjects, which resulted in different amino acid changes. However, the enzymatic activity of ALAD expressed by mutant ALAD was investigated only for 4 mutants, A274T and R240W, in German patient B, 3 and V153M and delTC in German patient H 11 (singleletter amino acid codes). This previous study with German patient B demonstrated that ALAD activity co...