ABSTRACT
Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.
Subject(s)
Fructose-1,6-Diphosphatase Deficiency/genetics , Fructose-Bisphosphatase/genetics , Point Mutation , Amino Acid Substitution , Base Sequence , Cells, Cultured , Child, Preschool , Female , Fructose-Bisphosphatase/metabolism , Genetic Carrier Screening , Humans , Infant , Infant, Newborn , Japan , Liver/enzymology , Male , Mutagenesis, Site-Directed , Pedigree , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Polymorphism, Single-Stranded ConformationalABSTRACT
The antisecretion effect of dl-(15R)-15 methyl-PGE2 methyl ester (PG6E) was studied in perfusing rats in vivo. The results showed that PG6E at the dose 0.1 mg.kg-1 decreased markedly the acid secretion and antagonized the gastric acid secretion induced by histamine, pilocarpin and pentagastrin in rats. This action of PG6E appeared to be similar to that of cimetidine (40 mg.kg-1).
Subject(s)
Arbaprostil/pharmacology , Gastric Acid/metabolism , Gastrointestinal Agents/pharmacology , Animals , Arbaprostil/analogs & derivatives , Histamine Antagonists , Male , Pentagastrin/antagonists & inhibitors , Pilocarpine/antagonists & inhibitors , RatsABSTRACT
The effects of dl-(15R)-15 methyl-PGE2 methyl ester (PG6E), in experimental ulcers induced by absolute alcohol, HCl, indomethacin, pyloric ligation and chronic acetic acid in rats were studied. PG6E at doses 10-80 micrograms.kg-1 was shown to have significant protective effect. PG6E (30-60 micrograms.kg-1) was also found to reduce markedly the acid secretion, pepsin activity, DNA content of the juice collected from pylorus ligated stomach of rats and increase markedly the content of gastric mucosa hexosamine in mice given PG6E (30-60 micrograms.kg-1 po for 3 d). At doses of 40-80 micrograms.kg-1, PG6E was able to have no significant effect on gastric emptying time in rats and gastrointestinal tract movement in mice. It appears that PG6E was shown to inhibit the aggressive factors and increase the protective factors of gastric mucosa. This may hopefully become a new antiulcer agent.
Subject(s)
Anti-Ulcer Agents/pharmacology , Arbaprostil/analogs & derivatives , Gastric Acid/metabolism , Stomach Ulcer/metabolism , Animals , Arbaprostil/pharmacology , Female , Gastric Emptying/drug effects , Gastrointestinal Motility/drug effects , Male , Mice , Rats , Rats, Wistar , Stomach Ulcer/physiopathologyABSTRACT
Fructose-1,6-bisphosphatase deficiency is an inheritable disorder of gluconeogenesis. Sequence analysis of the cDNA of the fructose-1,6-bisphosphatase mRNA isolated from monocytes from a girl with this disease and her consanguineous parents revealed that the patient and her parents were a homozygote and heterozygotes for an insertion of one G residue at G957GGGG961, respectively. This mutation resulted in translation of a truncated enzyme protein, and the mutant protein showed no fructose-1,6- bisphosphatase activity in an overexpression experiment in Escherichia coli. However, this mutation is located in a region of the amino acid sequence which is not well conserved among mammals. A mutagenized clone was prepared from the normal clone. The extents of substitutions and deletions of the amino acid sequence were predicted to be less in the mutagenized protein than in the mutant protein. This mutagenized clone also expressed no fructose-1,6-bisphosphatase activity, although both of two normal clones from control monocytes and a control liver sample expressed an apparently normal level of fructose-1,6-bisphosphatase activity. Thus, this mutation is concluded to be responsible for fructose-1,6-bisphosphatase deficiency in this patient.
Subject(s)
Fructose-1,6-Diphosphatase Deficiency/genetics , Fructose-Bisphosphatase/genetics , Mutation , Amino Acid Sequence , Base Sequence , Child , Consanguinity , DNA Primers , Female , Fructose-1,6-Diphosphatase Deficiency/enzymology , Genetic Carrier Screening , Homozygote , Humans , Male , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Family , Polymerase Chain Reaction/methods , RNA, Messenger/analysis , RNA, Messenger/biosynthesisABSTRACT
Fructose 1,6-bisphosphatase deficiency is an autosomal recessive inherited disorder of gluconeogenesis. We could isolate cDNAs encoding human fructose 1,6-bisphosphatase from normal monocytes, liver and kidney, but not from normal lymphocytes. The cDNAs contained an open reading frame coding for 338 amino acids, and their nucleotide sequences in monocytes and liver were identical. G644C645 nucleotides in this sequence were the same as those of cDNA from HL-60 cells, although our result differed from a previous report (M. El-Maghrabi et al. (1993) J. Biol. Chem. 268, 9466-9472) on an alteration to C644G645 nucleotides in human liver cDNA resulting in a change of Gly-214 to Ala-214 in the enzyme. The Gly-214 (GGC) residue was therefore conserved in the enzymes hitherto isolated from humans and other animals. Analysis of monocytes in seven patients with fructose 1,6-bisphosphatase deficiency showed a DNA fragment with apparent normal size in two sisters but no detectable DNA fragment in the other five patients. Monocytes were thus useful as an alternative source for mRNA from human liver for the molecular analysis of fructose 1,6-bisphosphatase deficiency.
Subject(s)
DNA, Complementary/chemistry , Fructose-1,6-Diphosphatase Deficiency/genetics , Fructose-Bisphosphatase/genetics , Glycine , Kidney/enzymology , Liver/enzymology , Monocytes/enzymology , Point Mutation , Alanine , Amino Acid Sequence , Base Sequence , Cell Line , DNA/genetics , DNA Primers , DNA, Complementary/metabolism , Fructose-Bisphosphatase/biosynthesis , Humans , Lymphocytes/enzymology , Molecular Sequence Data , Oligonucleotides, Antisense , Open Reading Frames , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Tumor Cells, CulturedABSTRACT
1-Carbobutoxy-beta-carboline, N9-formylharman, 1-carboethoxy-beta-carboline, 1-carbomethoxy-beta-carboline, perlolyrine, harman and norharman were isolated from the rhizoma of Polygala lenuifolia. The structures were elucidated on the basis of spectroscopic studies and chemical evidence, 1-carbobutoxy-beta-carboline has not been seported before.
