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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...

Although prostatic cancer is evident late in life, pathological evidence suggests this disease is initiated earlier in life. As prostatic cancer is an endocrine associated disease and as adult hormone profiles are established during puberty, it was of interest whether difference in pubertal hormone levels occurred in populations at low or high risk for prostatic cancer. Accordingly we have investigated the hormone profiles in rural Black South African and urban white boys during puberty. It has been suggested that the timing of puberty is modified by environmental factors and that there is a concomitant control of gonadotrophin release and food intake by CNS-peptide hormones. It is therefore postulated that dietary factors during puberty modify the gut-CNS peptide hormones which in turn control the hypothalamic-pituitarytesticular axis. Distinct difference in plasma androgen and gonadotrophins between the two races are in part concordant with a modification of CNS-peptide hormones by environmental factors during puberty.

Menstrual activity is dependent on a critical body weight and may, therefore, be modified by nutritional factors. Lower plasma levels of testosterone, androstenedione, dehydroepiandrosterone, and prolactin, and differences in gonadotrophin levels were found during the menstrual cycle in rural black South African versus white women. When premenopausal vegetarian South African black women were fed a Western diet, plasma testosterone and prolactin levels increased, while estradiol decreased and follicle-stimulative hormone increased. In postmenopausal black women a similar diet decreased plasma leutinizing and follicle-stimulating hormone and increased prolactin levels. Differences between white and black women eating their customary diets may be related to genetic factors and/or lifestyle and diet. However, a Western diet induced changes in hormonal activity in vegetarian black women. These changes in hormonal levels in black women were comparable to those found in women with menstrual irregularities. Further study is needed to determine whether the difference in hormonal activity during the menstrual cycle between vegetarian black and white women may in part explain the lower incidence of coronary heart disease and breast cancer in the former.

In Caucasian women, the length of the menstrual cycle is 28 days with a SD of 3.9 days. Many factors including diet modify menstrual periodicity. We have determined the effect of a meatless vegetarian diet in Caucasian women on the length of the menstrual cycle and the episodic and luteinizing releasing hormone-stimulated release of luteinizing hormone. The effect of a Western diet on the menstrual cycle in urban Black South African women was also studied. In Caucasian women, the vegetarian diet decreased (p less than or equal to 0.01) the pituitary response to luteinizing releasing hormone and decreased (p less than or equal to 0.05) the episodic release of luteinizing hormone. A Western diet increased (p less than or equal to 0.01) the duration of the follicular phase in Black South African women. Thus diet is a factor modifying the episodic release of gonadotrophins and follicular maturation.

Cloning, expression, and genotype studies of the defective gene for δ-aminolevulinate dehydratase (ALAD) in a patient with an unusual late onset of ALAD deficiency porphyria (ADP) were carried out. This patient was unique in that he developed the inherited disease, together with polycythemia, at the age of 63. ALAD activity in erythrocytes of the patient was less than 1% of the normal control level. ALAD complementary DNA (cDNA) isolated from the patient's Epstein-Barr virus (EBV)–transformed lymphoblastoid cells had 2 base transitions in the same allele, G177 to C and G397 to A, resulting in amino acid substitutions K59N and G133R, respectively. It has been verified that the patient had no other ALAD mutations in this and in the other allele. By restriction fragment length polymorphism (RFLP) analysis, all family members of the proband who had one-half ALAD activity compared with the ALAD activity of the healthy control were shown to have the same set of base transitions. Expression of ALAD cDNA in CHO cells revealed that K59N cDNA produced a protein with normal ALAD activity, while G133R and K59N/G133R cDNA produced proteins with 8% and 16% ALAD activity, respectively, compared with that expressed by the wild type cDNA. These findings indicate that while the proband was heterozygous for ALAD deficiency, the G397 to A transition resulting in the G133R substitution is responsible for ADP, and the clinical porphyria developed presumably due to an expansion of the polycythemic clone in erythrocytes that carried the mutant aladallele.

The level of mRNA encoding beta-globin was examined in dimethyl sulfoxide (DMSO)-sensitive (DS), and DMSO-resistant (DR) murine erythroleukemia (MEL) cells. DR cells lack erythroid-specific delta- aminolevulinate (ALA) synthase (AL-AS-E), and fail to undergo erythroid differentiation following treatment with DMSO. Treatment of cells with DMSO markedly increased ALAS-E mRNA in DS cells, while the same treatment downregulated the nonspecific ALA synthase (ALAS-N) mRNA levels in both DS and DR cells. The levels of beta-globin mRNA, heme content, and hemoglobin in DS cells increased, while those in DR cells decreased following treatment with DMSO. Treatment of DR cells with hemin caused an increase in beta-globin mRNA and hemoglobin, and partially restored the DMSO-mediated suppression of beta-globin mRNA and hemoglobin synthesis. DMSO treatment decreased heme oxygenase (HO) mRNA in hemin-treated DS cells, but not in hemin-treated DR cells. These findings indicate that heme is necessary for accumulation of the beta-globin transcript during erythroid differentiation, and that hemin- mediated HO induction becomes markedly downregulated in differentiated erythroid cells, presumably because less free heme is available for HO induction by a greater demand for the synthesis of hemoglobin.

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