mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise.

A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.

Adenoviral-mediated expression of porphobilinogen deaminase in liver restores the metabolic defect in a mouse model of acute intermittent porphyria.

The aim of this study was to investigate the potential of gene therapy in the treatment of acute intermittent porphyria (AIP), a disorder caused by a partial deficiency of porphobilinogen deaminase (PBGD), the third enzyme in heme synthesis. The condition is biochemically characterized by accumulation of the porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here we present the first experiments in vivo using adenoviral vectors to replace the deficient enzyme in the liver of an AIP mouse model. The use of adenoviral vector carrying the cDNA of luciferase in wild-type mice confirmed that transgene expression after intravenous administration was found mainly in liver. When PBGD-deficient mice were administered with adenoviral vector carrying the cDNA of mouse PBGD, the hepatic PBGD activity increased in a dose- and time-dependent manner. The highest activity was found 7 days after injection and remained high after 29 days. The expressed enzyme was shown to correct the metabolic defect in the PBGD-deficient mice as no accumulation of ALA or PBG occurred in plasma, liver, or kidney after induction of heme synthesis by phenobarbital. The study demonstrates that hepatic PBGD expression prevents the accumulation of porphyrin precursors, suggesting a future potential for gene therapy in AIP.

Enzymes for heme biosynthesis are found in both the mitochondrion and plastid of the malaria parasite Plasmodium falciparum.

All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum. We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway delta-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg2+ (and enhanced by Mn2+) but not by Zn2+. A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.

Non-viral delivery of the porphobilinogen deaminase cDNA into a mouse model of acute intermittent porphyria.

Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deficient activity of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD). Clinical symptoms of this autosomal dominant hepatic porphyria include episodic acute attacks of abdominal pain, neuropathy, and psychiatric disturbances. Current therapy based on intravenous heme administration is palliative and there is no way to prevent the attacks. Thus, efforts are focused on methods to replace the deficient activity in the liver to prevent the acute attacks of this hepatic porphyria. Here we explore the efficiency of a non-viral gene delivery to obtain PBGD expression in the liver of AIP transgenic mice. Four vectors were evaluated: naked DNA and DNA complexed to liposomes, polyethylenimine (PEI), and PEI-galactose, using a luciferase construct as reporter gene. The vectors were administered intravenously or directly into the portal vein with transient blood flow blockage. After tail vein injection of the DNA complexes, the liposome vector had the highest luciferase expression in lung and less in liver. When injected into the portal vein, the naked DNA had considerably higher hepatic reporter gene expression; 100 microg of naked DNA had the highest hepatic luciferase expression 24h after portal vein injection. When these vectors were used to deliver the PBGD gene into the AIP mouse model no enhancement of the endogenous PBGD activity in liver was detectable, despite the presence of the PBGD-plasmids as verified by PCR. Thus, more efficient non-viral vectors are needed to express sufficient PBGD activity over the endogenous hepatic level (approximately 30% of normal) in this murine system.

Non-viral mediated gene transfer of porphobilinogen deaminase into mammalian cells.

Acute intermittent porphyria (AIP) is an inborn error of heme synthesis in which the third enzyme, porphobilinogen deaminase (PBGD), is deficient. The disease is characterized by recurrent attacks of acute abdominal pain often accompanied by neuropsychiatric symptoms. Current therapeutic treatment with heme is only palliative and no curative alternative exists. The present report describes the first step towards a gene therapy treatment for AIP. Mouse cDNA encoding the PBGD enzyme was cloned and four vectors containing the full-length mouse and human cDNA of the housekeeping and erythroid PBGD isoforms under the control of a cytomegalovirus promoter were constructed. The vectors, condensed to polyethylenimine, were successfully transfected to NIH 3T3 and HeLa cells as determined by enzymatic activity measurements. Thus, the PBGD activity was increased 3-10 times in NIH 3T3 cells and 95-240 times in HeLa cells. The expression was shown to be dose and time dependent, with the highest level of activity observed in HeLa cells after 72 h posttransfection. Non-viral gene transfer was also undertaken in PBGD-deficient fibroblasts established from an AIP patient. Complete normalization of the PBGD activity was accomplished after the addition of 2.5 microg DNA per well. Further addition of DNA increased the PBGD activity up to threefold the normal value. The study documents a successful gene transfer and a high degree of PBGD expression in different cell-lines, indicating a potential for future gene therapy in AIP.

Plastid state- and light-dependent regulation of the expression of nucleus-encoded genes for chloroplast proteins in the flagellate Euglena gracilis.

Interorganellar regulatory interactions in the flagellate Euglena gracilis were shown to be more complicated than in green algae and higher plants. Euglena plastids have a much more complex influence on nuclear gene expression than was previously thought. The petJ gene for cytochrome c6 represents a group of nucleus-encoded genes for chloroplast proteins, the expression of which is influenced by the state of plastids at the transcriptional level. Moroever, the regulation of these genes might be light-dependent. In contrast, for nucleus-encoded small subunit of ribulose-bisphosphate carboxylase, chlorophyll a/b binding protein, and porphobilinogen deaminase transcript levels are unchanged in wild-type cells relative to white mutants. In these cases there is no plastid-derived signal operative during transcription. Porphobilinogen deaminase appeared to be regulated even at the post-translational level.

Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR.

We used a recently developed system for real-time quantitative polymerase chain reaction (PCR) to determine residual disease in patients with chronic myeloid leukaemia. The expression of the Bcr-Abl hybrid oncogene was determined and normalized by using the PBGD housekeeping gene product as endogenous reference. The sensitivity and reproducibility of the assay was tested on cell line K562. A dilution of Bcr-Abl-positive cell line K562 remained positive at up to 250 fg of RNA. 10 copies of Bcr-Abl DNA in water could still be detected. The dynamic range of the method spanned six orders of magnitude. Analysis of 10 identical assays on K562 RNA resulted in a variation of 15%. To test the feasibility of normalization of Bcr-Abl dosage by the PBGD product, we compared the efficiencies of the RT-PCRs in 150 patient analyses. We concluded that PBGD was a suitable and stringent quality control standard. Three patients who were treated with donor leucocyte infusions for chronic myeloid leukaemia who had relapsed after bone marrow transplantation were followed over time. The normalized Bcr-Abl dosage was compared to the results of cytogenetics. Cytogenetic analysis was negative below a normalized Bcr-Abl dose of about 3 x 10(-2). This semi-automated method is fast, sensitive and accurate and enables a high throughput of samples.

Cloning and sequencing of a previously unidentified gene that is involved in the biosynthesis of heme in Escherichia coli.

We have isolated a number of porphyrin (Por)-synthesis mutants as light-resistant revertants of a light-sensitive strain delta visA (hemH) of Escherichia coli that accumulates protoPor IX in the cell. Among such mutants, we found a double mutant (H103) with mutations in hemA and in a new gene downstream of hemA. This new gene, designated hemK, was located at 27 min on the linkage map of the E. coli chromosome. By nucleotide (nt) sequencing, it was demonstrated that hemK forms part of the hemA-prfA-hemK operon and encodes 225 amino acids that show no significant homology to any protein in the standard databases. The mutant strain H103 formed small colonies and showed no catalase activity even in the presence of 5-aminolevulinic acid (ALA), indicating its inability to catalyze a step in the biosynthesis of heme from ALA. An extract of H103 cells has readily detectable ALA dehydratase and porphobilinogen deaminase activities. H103 cells carrying a plasmid that included only hemA as an insert accumulated protoPor and coproPor, but showed no sensitivity to light, a result that suggests that it may be deficient in protoporphyrinogen oxidase activity.

Yeast porphobilinogen deaminase also forms enzyme-pyrrole intermediates.

The enzyme porphobilinogen deaminase (PBG deaminase, EC 4.3.1.8) catalyzes the condensation of four molecules of PBG to give the linear tetrapyrrol, hydroxymethylbilane. It has been shown that this enzyme forms stable mono-, di-, tri- and tetrapyrrole-enzyme covalent complexes. When the enzyme, partially purified in the absence or presence of phenylmethylsulfonyl fluoride (PMSF) and preincubated with PBG, was applied on DEAE-cellulose columns, three peaks with PBG deaminase activity were detected. Using Ehrlich's reagent, it was found that the active peaks corresponded to mono-, di- and tri-pyrrylmethane-enzyme complexes. Therefore, the mechanism of action of PBG deaminase from Saccharomyces cerevisiae also involves the sequential addition of four PBG units, leading to the formation of the enzyme-substrate intermediate complexes, as has already been described for the same enzyme from other sources.

The role of ALA-S and ALA-D in regulating porphyrin biosynthesis in a normal and a HEM R+ mutant strain of Saccharomyces cerevisiae.

Catabolite repression and derepression on delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) in a normal yeast strain, D27, and its derived D27/C6 (HEM R+) were investigated. ALA-S and ALA-D activities and intracellular ALA (I-ALA) at different physiological states of the cells were measured. In YPD medium, under conditions of repression and when glucose was exhausted, both strains behaved identically as if the mutation was not expressed. In YPEt medium, however, both ALA-S and ALA-D activities were higher than in YPD, but the I-ALA content and the enzymic activity profiles shown by the two strains were quite different. It appears, therefore, that the mutation causes a deregulation of ALA-S, so that its activity is kept at a high level throughout the cell cycle. This would explain the increased levels of cytochromes present in the mutant. This mutation may affect some regulatory aspect of ALA formation and renders an ALA-S of high activity; moreover, this enzyme species seems to be more stable than in the normal strain.

Selective accumulation of endogenously produced porphyrins in a liver metastasis model in rats.

The possibility of using the porphyrin precursor 5-aminolevulinic acid to cause selective porphyrin accumulation in tumors was examined. Syngeneic colon carcinomas CC531 were implanted in the livers of Wag/Rij rats. Groups of three to six animals each were given 2 mg/mL of 5-aminolevulinic acid in drinking water from the 8th, 14th, or 17th day after tumor implantation. Two other groups received either 2.5 or 5 mg/kg of Photofrin II (Photomedica Inc., Raritan, NJ) intravenously on day 17. On day 19 the livers were removed and porphyrin concentrations were measured in normal livers and tumors by solvent extraction and high-performance liquid chromatography. Protoporphyrin accumulated progressively in tumors with increasing duration of 5-aminolevulinic acid administration (P = 0.0001), whereas no increase was found in normal livers. After 11 days of 5-aminolevulinic acid administration the porphyrin concentration ratio between tumors and livers was 4:1. In contrast, after Photofrin II administration the concentration was higher in normal livers than in tumors (1:3 ratio, tumor to liver). Enzyme measurements showed a threefold decrease in ferrochelatase activity in tumors compared with livers (P less than 0.001). In conclusion, oral administration of 5-aminolevulinic acid results in progressive accumulation of protoporphyrin in a transplantable colon carcinoma without accumulation in the surrounding liver tissue. This selective accumulation of porphyrins appears to be caused by a relative ferrochelatase deficiency in malignant tissue. 5-Aminolevulinic acid administration may be a suitable approach to photosensitizing liver tumors for photodynamic therapy or to early detection of tumors by fluorescence in ultraviolet light.

Porphyrin metabolism in some malignant diseases.

Porphyrin metabolism was studied in 21 children of both sexes suffering from acute lymphoblastic leukaemia (ALL) and 34 adult patients of different ages and sexes suffering from ALL (n = 14), non-Hodgkin's lymphoma (NHL), n = 14, or Hodgkin's disease (HD), n = 6. In addition, two groups of healthy children (n = 14), and adults (n = 17) were studied for comparison. It was apparent from this study that the activity of uroporphyrinogen-1-synthetase (URO-1-S, E.C. 4.3.1.8) was highly significantly activated in the blood of children, while the activities of blood 5-aminolevulinic acid dehydrase (E.C. 4.2.1.24) and ferrochelatase (E.C. 4.99.1.1.), as expressed by protoporphyrin/haem ratio, were inhibited in those children. Also, free erythrocyte total porphyrins were increased, while the haem content was reduced. The concentrations of 5-aminolevulinic acid, coproporphyprin and uroporphyrin were increased in the urine of children with ALL. On the other hand, some dramatic changes were found in porphyrin metabolism in adult patients suffering from ALL, NHL and HD. The aforementioned disturbances were discussed in the light of some factors which may affect the enzymatic activities in the synthesis of porphyrins.

Accelerated heme synthesis and degradation in transformed fibroblasts.

Various parameters of the heme biosynthetic pathway were studied in two cell lines, one nontransformed and the other malignantly transformed (MLV/MS), both replicating at the same rate. Using the above system enabled us to distinguish between phenomena characteristic of the malignant transformation per se and those due to accelerated growth rate. Heme synthesis and degradation as well as the activities of ALAS, ALAD, PBGD, and FC were found to be increased in the transformed cells. However, the concentration of intracellular heme was markedly reduced from 30.4 +/- 4.4 pmole/mg protein in nontransformed cells to 10.5 +/- 2.6 pmole/mg protein in transformed cells. These observations show that malignant transformation leads to changes in heme metabolism unrelated to growth rate in this cell line.

[Do not forget: lead poisoning]. / Nicht vergessen: Bleivergiftung.

3 patients with lead intoxication are demonstrated. The analysis of porphyrins and porphyrin precursors led to the tentative diagnosis which was confirmed by the increased lead levels. The increased urinary excretion of porphyrins and of delta-aminolevulinic acid, the decreased activity of uroporphyrinogen synthase and the increased protoporphyrin content (absorption maximum 595 nm) in the red cells suggested the diagnosis before the lead values of the patients were known. Clinical signs of the patients included gastrointestinal and neurological symptoms. It is assumed that the lead intoxication in these patients was caused by occupational exposition.