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Biomedical subjects

S Sassa

Publications and source records attributed to S Sassa.

At least 55 records · Page 3Linked to original sources
PubMed1997

Molecular basis of variegate porphyria: a de novo insertion mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders that result from the inherited or acquired dysregulation of one of the eight enzymes in the heme biosynthetic pathway. Variegate porphyria (VP) is characterized by deficiencies in protoporphyrinogen oxidase (PPO) and has recently been genetically linked (Z = 6.62) to the PPO gene on chromosome 1q21. In this study, we have identified two sequence variants in the PPO gene in a family with VP. The first is a neutral polymorphism at the -47 position of intron 2; this polymorphism is present in the general population and is unlikely to underlie the VP phenotype. The second is a mutation in the PPO gene in a patient with VP; the mutation consists of an apparently de novo 2-bp insertion in exon 3 of PPO and results in a frameshift and downstream premature termination codon. These data establish that a frameshift mutation in PPO is the underlying mutation in this patient with VP and explain the sporadic occurrence of the phenotype in this family.

Adult↗
PubMed1997

5-Aminolevulinate synthase expression and hemoglobin synthesis in a human myelogenous leukemia cell line.

We examined the effect of hemin, TGF-beta1 and cytosine arabinoside (Ara-C) on the levels of mRNAs for the erythroid-specific 5-aminolevulinate synthase (ALAS-E) and gamma-globin in various human myelogenous leukemia cell lines. Detailed analyses were also made using one of them, YN-1, which was isolated and established in culture from a patient with chronic myelogenous leukemia. Our results demonstrate that gamma-globin protein level and the percentage of benzidine-positive cells in the cell line increased markedly (10- to 30-fold) upon treatment with hemin, TGF-beta1, or Ara-C. In contrast, gamma-globin mRNA was already markedly expressed prior to treatment in 4 out of 9 cell lines examined, including YN-1, and the level increased only marginally after treatment with hemin. ALAS-E mRNA levels were increased in YN-1 cells after treatment with TGF-beta1 and Ara-C, while hemin treatment had little effect. These results indicate that heme supply is insufficient in YN-1 cells and suggest that hemin increases hemoglobin synthesis principally at the post-transcriptional level, whereas TGF-beta1 and Ara-C stimulate hemoglobin synthesis by activating efficient endogenous heme supply in the cells.

5-Aminolevulinate Synthetase↗
PubMed1997

Lead binding to delta-aminolevulinic acid dehydratase (ALAD) in human erythrocytes.

Over 99% of the lead present in blood is usually found in erythrocytes. To investigate the nature of this selective accumulation of lead in erythrocytes, the specific binding of lead to proteins in human erythrocytes was studied using liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS). The principal lead-binding protein had a mass of approximately 240 kDa, and adsorption to specific antibodies showed that protein was delta-aminolevulinic acid dehydratase (ALAD). Thus, the previous notion that lead in erythrocytes was bound primarily to haemoglobin has to be revised. Furthermore, in lead-exposed workers, the percentage of lead bound to ALAD was influenced by a common polymorphism in the ALAD gene. Specifically, in seven carriers of the ALAD2 allele, 84% of the protein-bound lead recovered was bound to ALAD compared to 81% in seven homozygotes for the ALAD1 allele whose erythrocytes were matched for blood-lead concentration. The small difference was statistically significant in Wilcoxon matched-pairs signed-rank test (P = 0.03). No ALAD allele-specific difference in ALAD-bound lead was found among 20 unexposed controls. Perhaps the difference in ALAD-bound lead can provide an explanation for the previously reported finding of higher blood-lead levels among carriers of the ALAD2 allele than among ALAD1 homozygotes in lead-exposed populations.

Adult↗
PubMed1997

Effects of conjugated estrogens with or without medroxyprogesterone acetate on mammary carcinogenesis, uterine adenomyosis and femur in mice.

Although combined hormone replacement therapy using estrogens and progestins reduces the risk of endometrial cancer in postmenopausal women, it has been unclear whether the combined therapy is associated with an alteration of the risk of mammary cancer. Virgin mice of the SHN strain, which has a high potential for the development of mammary cancer and uterine adenomyosis, were given diets containing conjugated estrogens with or without medroxyprogesterone acetate for 230 days. The combined administration of conjugated estrogens and medroxyprogesterone acetate completely suppressed the development of uterine adenomyosis with a decrease in uterine thymidylate synthetase activity, significantly enhanced the bone mineral density in the femur and slightly shortened the latent period of mammary carcinogenesis.

Adenoma↗
PubMed1997

Molecular defects of the coproporphyrinogen oxidase gene in hereditary coproporphyria.

Hereditary coproporphyria (HCP) is an acute hepatic porphyria, and is an autosomal dominant disorder but with a variable degree of clinical expression. Molecular cloning, sequencing and expression of the defective gene for coproporphyrinogen oxidase (CPO) in a patient with HCP were carried out. Enzyme assays revealed that CPO activity in EBV-transformed lymphoblastoid cells from the proband and one of her sisters was approximately 50% of normal. Nucleotide sequence analysis of CPO cDNAs isolated from the proband's cells demonstrated 3 base substitutions which accompanied 3 different amino acid substitutions. An A514-->C transition causing an Asn172-->His substitution occurred in one allele, while two other transitions, G265-->A and G580-->A, caused Gly89-->Ser and Val194-->Ile substitutions, respectively, in the other allele. The A514-->C and the G580-->A transitions were shown to be genetic polymorphisms. Transfection of CPO cDNA into E. coli demonstrated that cDNA with the G265-->A transition produced a protein with less than 5% of normal enzyme activity. These findings indicate that the G265-->A transition, involving the highly conserved glycine residue at the 89th position, is responsible for the CPO defect in the patient and accounts for the partial deficiency of CPO activity in this pedigree. This mutation is different from three other mutations reported in patients with HCP. Molecular defects in the porphyrias including HCP are highly heterogeneous.

Cell Line, Transformed↗
PubMed1997

Preventive effects of polysaccharides extracted from human tubercle bacilli (specific substance of Maruyama) on colonic carcinogenesis in rats.

Polysaccharides extracted from human tubercle bacilli (specific substance of Maruyama [SSM]) have been clinically applied with satisfactory results. Thymidylate synthetase (TS) and thymidine kinase (TK) are key enzymes in de novo and salvage pathways for pyrimidine nucleotide synthesis. Well- and moderately well differentiated adenocarcinomas induced with 1,2-dimethylhydrazine (DMH) are widely distributed throughout the colorectal tract with high TK activity, and the poorly differentiated type is mainly restricted in the proximal colon and the cecum with high TS activity in rats. Subcutaneously injecting the rats with SSM reduced TS activity in colonic nontumorous regions, but in the tumorous regions it reduced TK activity compared with that of the DMH-treated rats without SSM treatment. SSM is suggested to reduce the colorectal carcinogenesis induced with DMH by inhibiting DNA synthesis in a de novo pathway, and to suppress the development of the tumors by decreasing DNA synthesis in the salvage pathway in rats.

1,2-Dimethylhydrazine↗
PubMed1996

Five new mutations in the uroporphyrinogen decarboxylase gene identified in families with cutaneous porphyria.

We describe five new mutations in the uroporphyrinogen decarboxylase (UROD) gene. All mutations were observed in conjunction with decreased erythrocyte UROD and clinical familial porphyria cutanea tarda (fPCT), (four families) or hepatoerythropoietic porphyria (HEP), (one family). The fPCT mutations included three point mutations that resulted in amino acid substitutions: a lysine to glutamine at amino acid position 253 (exon 7); a glycine to arginine at position 318 (exon 10); an isoleucine to threonine at position 334 (exon 10). The lysine to glutamine at amino acid position 253 was found in conjunction with a single C nucleotide deletion in exon 8 on the same allele of the UROD gene in the same family. This deletion resulted in a shift in the reading frame and the introduction of a premature stop codon 8 amino acids downstream. In the fourth family, a 31-bp deletion (nucleotides 828-858: exon 8) of the coding region, resulted in a frameshift and the introduction of a stop codon 19 amino acids downstream. A point mutation was observed in an individual diagnosed with HEP, resulting in an alanine to glycine change at amino acid position 80 and was present on both alleles. All mutations were confirmed in at least one other family member. The impact of these mutations on the function of the UROD protein was examined using in vitro protein expression and with activity assessed using pentacarboxylic acid porphyrinogen I as a substrate for UROD. Although three mutations reduced UROD activity to < 15% of normal, one resulted in a UROD protein with 50% functional activity and the other had near normal activity. These results indicate that many different genetic lesions of the UROD gene are associated with fPCT.

Family↗
PubMed1996

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Animals↗
PubMed1996

Chronic effect of hyperprolactinemia on blood glucose and lipid levels in mice.

We studied the chronic effects of hyperprolactinemia, induced by ectopic pituitary grafting, on blood glucose and lipid levels in adult male mice. For one year after pituitary grafting, we measured the blood levels of prolactin, growth hormone (GH), insulin, glucose and free fatty acid (FFA) at various intervals. The graft caused consistent hyperprolactinemia without changes in the serum GH levels. Hypoglycemia developed at 1 and 3 months after grafting but was not accompanied by any changes of the serum insulin levels. Thereafter, the blood glucose and serum insulin levels began to increase in the pituitary-grafted (PG) mice, and at 12 months after the operation, both levels became significantly higher in PG mice than controls. The serum FFA levels and the weight of epididymal fat bodies were significantly lower in PG mice than controls from 3-12 months after the grafting. Thus, hyperprolactinemia leads to persistent hypolipidemia and biphasic changes in the blood glucose level.

Adipose Tissue↗
PubMed1996

The role of heme in gene expression.

Recent evidence indicates that there are significant tissue-specific expression and heme-mediated regulation of enzymes in the heme biosynthetic pathway. In particular, delta-aminolevulinate synthase (ALAS) [EC 2.3.1.37] has been shown to exist as tissue-specific isozymes, i.e. the erythroid and non-specific ALAS, which are coded by two separate genes. In mammals, ALAS activity can be found in many tissues, the highest activity being found in the Harderian gland in rodents, the liver of chemically induced porphyric animals, and developing erythroblasts. In the liver, ALAS expression is under negative control by heme, while in the developing erythroblasts it is positively influenced by heme. In contrast to these tissues, ALAS is maximally expressed in the Harderian gland and is not influenced by heme. In addition to the tissue-specific regulation of heme biosynthesis, heme has also been shown to influence a number of gene functions that are not directly related to heme synthesis in various tissues.

5-Aminolevulinate Synthetase↗
PubMed1996

Diagnosis and therapy of acute intermittent porphyria.

The porphyrias are a group of disorders caused by deficiencies in the activities of the enzymes of the heme biosynthetic pathway. As a result, abnormally elevated levels of porphyrins and/or their precursors, e.g. delta-aminolevulinic acid and porphobilinogen are produced in excess, accumulate in tissues, and are excreted in urine and stool. Two cardinal symptoms of the porphyrias are cutaneous photosensitivity and neurologic disturbances. Acute intermittent porphyria is the most important form of hepatic porphyria because of its frequency and severe clinical symptoms.

Humans↗
PubMed1996

1-(2-Tetrahydrofuryl)-5-fluorouracil in combination with uracil suppresses mammary carcinogenesis and growth of tumors induced with 7,12-dimethylbenz[a]anthracene in rats.

The effects of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil (UFT) on mammary carcinogenesis and growth of tumors induced with 7,12-dimethylbenz[a]anthracene (DMBA) were investigated in rats. Daily oral administration of UFT reduced the incidence and number of mammary tumors compared with those of the DMBA control group, resulting in lower activities in DNA synthesizing enzymes, thymidylate synthetase and thymidine kinase, and a reduction of bromodeoxyuridine-immunoreactive (S-phase) cells in mammary tumors of UFT-treated rats.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Pharmacotherapeutic effects of toki-shakuyaku-san on leukorrhagia in young women.

Toki-shakuyaku-san is a traditional Chinese herbal prescriptions that is composed of 6 herbal plants, i.e., peony root, atractylodes lancea rhizome, alisma rhizome, hoelen, cnidium rhizome and Japanese angelica root. Administration with Toki-shakuyaku-san normalized irregular menstrual cycle, healed cervical pseudo-erosion and reduced leukorrhagia in young women who had insufficient luteal function.

Adult↗
PubMed1996

Cimetidine in the treatment of porphyria cutanea tarda.

The efficacy of the H2 receptor antagonist, cimetidine, in the treatment of a patient with porphyria cutanea tarda (PCT) was evaluated. After administration of cimetidine for 2 weeks, urinary excretion of uroporphyrin (UP) and coproporphyrin (CP) was significantly decreased. Urinary porphyrin levels remained low even after the cessation of cimetidine for 1 week. Although the readministration of cimetidine did not decrease porphyrin excretion, skin lesions were markedly improved, and serum gamma-glutamyl transpeptidase (GGT), aminotransferases and serum ferritin decreased to the normal range. These results suggest that, in addition to efficacy in the treatment of acute intermittent porphyria (AIP) and erythropoietic protoporphyria (EPP), cimetidine is effective in the treatment of PCT.

Cimetidine↗
PubMed1996

Abnormal serum porphyrin levels in patients with the acquired immunodeficiency syndrome with or without hepatitis C virus infection.

OBJECTIVE: To define the contributions of human immunodeficiency virus (HIV) and hepatitis C virus infections to the development of porphyria cutanea tarda. DESIGN: Analysis of serum porphyrin levels in a cohort of 167 subjects. Serum samples were divided into 4 groups corresponding to the status of HIV and hepatitis C virus infections: positive-positive, positive-negative, negative-positive, and negative-negative. SETTING: Serum samples positive for HIV were obtained from the serum bank of an acquired immunodeficiency syndrome-HIV research center, and HIV-negative samples were obtained from a regional blood center. MAIN OUTCOME MEASURES: Spectrofluorometric measurement of serum porphyrin levels. RESULTS: The median values of porphyrin were 2.31 nmol/L (interquartile range [difference between the 25th and 75th percentiles]: 4.55) in the positive-positive group, 1.99 nmol/L (interquartile range: 1.63) in the positive-negative group, 1.31 nmol/L (interquartile range: 1.58) in the negative-positive group, and 1.14 nmol/L (interquartile range: 0.92) in the negative-negative group. The fluorescence emission spectra of samples with elevated porphyrin levels were identical with that reported for porphyria cutanea tarda. Elevated porphyrin levels were significantly associated with HIV infection (P < .001) and were observed in patients with an elevated level of alanine aminotransferase (P = .03). Infection with hepatitis C virus was also associated with an elevation in porphyrin levels, although the increase was not statistically significant (P = .16). Porphyrin levels in patients positive for HIV were not correlated with serum urea nitrogen or creatinine levels. None of the patients had symptomatic porphyria cutanea tarda. CONCLUSIONS: Factors associated with increased serum porphyrin levels included HIV infection, elevated alanine aminotransferase levels, and, to a lesser extent, hepatitis C virus infection. These findings suggest that patients with the above risk factors are potentially predisposed to the development of symptomatic porphyria cutanea tarda on further exposure to hepatotoxic agents.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Amino Acid Sequence↗
PubMed1995

The role of the erythroid-specific delta-aminolevulinate synthase gene expression in erythroid heme synthesis.

Using antisense technology, the effects of suppressed gene expression of the erythroid-specific delta-aminolevulinate (ALA) synthase (ALAS-E) on heme synthesis, expression of mRNAs encoding an erythroid-specific transcription factor NF-E2, other heme pathway enzymes, and beta-globin were examined in murine erythroleukemia (MEL) cells. In MEL cells in which an antisense ALAS-E RNA was expressed (AS clone), sense ALAS-E mRNA levels in both untreated and dimethylsulfoxide (DMSO)-treated cells were decreased compared with their respective controls. Heme synthesis in AS clones was decreased in proportion to the suppressed levels of ALAS-E mRNA. In addition, mRNAs for ALA dehydratase, porphobilinogen deaminase, ferrochelatase (FeC), and beta-globin were also decreased in AS clones. There was a strong correlation between the level of ALAS-E mRNA and most of the mRNAs of the heme pathway enzymes and beta-globin. There was a decrease in the mRNA level of p45, but not of mafK, which are the large and the small subunits of NF-E2, respectively, in AS clones. Treatment of AS cells with hemin and ALA in the presence of DMSO partially restored the suppressed mRNA levels for beta-globin and FeC and heme content, respectively. These findings thus indicate that heme formation, which is determined by the level of ALAS-E, plays an essential role on gene expression of many proteins necessary for erythroid development.

5-Aminolevulinate Synthetase↗