ABSTRACT
Hypophosphatasia is a rare disease characterized by low serum levels of tissue non-specific alkaline phosphatase (TNSALP) and a spectrum of skeletal disease varying from the severest form with death in utero to mild with no clinical abnormality in adults. Currently, the diagnosis of hypophosphatasia is made on the basis of clinical findings, radiography, low serum alkaline phosphatase levels and raised abnormal phosphorylated metabolites; there are elevations in serum pyridoxal 5'-phosphate, urinary phosphoethanolamine and inorganic pyrophosphate. In borderline cases the biochemical diagnosis remains uncertain. Prenatally, diagnosis is made using radiography and ultrasonography together with chorionic villus tissue biopsy, in which TNSALP levels are measured using an antibody-based assay. Since hypophosphatasia results from mutations in the TNSALP gene we have, for the first time in two U.K. families, undertaken restriction fragment length polymorphism (RFLP) analysis using three intragenic RFLPs for BclI and MspI at the ALPL locus. One family was informative, and a mutant-allele-specific haplotype with respect to three RFLPs was defined. In the other family the disease was shown to segregate with one allele of the BclI RFLP, but the MspI RFLPs were not informative. The disease segregated in the two families with different alleles of the BclI RFLP, suggesting that the mutations are likely to be different. We confirm that DNA analysis is likely to be the way ahead for diagnosing hypophosphatasia, and that standardized screening methods need to be developed for detecting mutations in these and other families.
Subject(s)
Alkaline Phosphatase/genetics , Hypophosphatasia/diagnosis , Adult , Age of Onset , Aged , DNA/analysis , Female , Genetic Markers , Humans , Hypophosphatasia/genetics , Infant , Male , Middle Aged , Mutation/genetics , Pedigree , Polymorphism, Restriction Fragment LengthSubject(s)
Blindness/genetics , Genetic Carrier Screening , Adult , Cataract/genetics , Child , Child, Preschool , Female , Genetic Linkage , Humans , Male , Pedigree , Retinal Detachment/genetics , X ChromosomeSubject(s)
Cystic Fibrosis/genetics , DNA/genetics , Electrophoresis, Agar Gel/methods , Alleles , Biotechnology , Costs and Cost Analysis , DNA/isolation & purification , Electrophoresis, Agar Gel/economics , Evaluation Studies as Topic , Female , Genetic Techniques , Humans , Male , Mutation , PedigreeSubject(s)
Sex Chromosome Aberrations/diagnosis , X Chromosome , Adult , Autoradiography , Humans , Male , Polymorphism, Restriction Fragment Length , SyndromeABSTRACT
The fes mutation in Escherichia coli K12, which inactivates enterochelin esterase, allows the cell to accumulate ferric enterochelin. The ferric complex of enterochelin was released in significant quantities from a fes mutant after osmotic shock. Analysis of the effects of the individual stages of the shock procedure in wild-type cells showed that prior exposure of cells to sucrose and EDTA was not required, careful dilution of cells into a hypo-osmolar medium being sufficient to induce efflux of Fe3+. Prior treatment with EDTA or exposure to shearing forces served either to enhance efflux or to induce efflux in isotonic media. Neither vitamin B12 nor 5'-nucleotidase was released from the periplasm by these procedures. The release observed under mild conditions was stimulated specifically by Co2+, did not occur at 0 degree C, and was inhibited by 2,4-dinitrophenol at 37 degrees C. From these observations, it was concluded that the efflux of Fe3+ represents a physiological response of the cell to exposure to a hypo-osmolar medium. Such changes may enhance survival following physicochemical stressing of the bacterial outer membrane.
Subject(s)
Enterobactin/metabolism , Escherichia coli/metabolism , Serine/analogs & derivatives , Biological Transport , Cations/metabolism , Iron Radioisotopes , Osmotic Pressure , Radioisotope Dilution TechniqueABSTRACT
The scandium complex of enterochelin is known to be assimilated and degraded by enterobacterial pathogens, liberating intracellular scandium. Macromolecular synthesis was inhibited in the order RNA, protein, DNA and phospholipid. Some component of heat-inactivated serum acts in concert with the complex inducing RNA degradation and killing. Both processes are inhibited by dinitrophenol. These biochemical changes resemble those found during complement mediated killing. An examination of the effect on synchronously growing cells suggests that the complex may exert its primary effect during the initiation of chromosome replication.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Enterobactin/administration & dosage , Scandium/administration & dosage , Serine/analogs & derivatives , Bacterial Proteins/biosynthesis , Cell Survival/drug effects , DNA, Bacterial/biosynthesis , Klebsiella pneumoniae/growth & development , Klebsiella pneumoniae/metabolism , RNA, Bacterial/biosynthesisABSTRACT
There is good evidence to show that ferric enterochelin is an essential growth factor for a number of Gram-negative pathogenic bacteria exposed to the host iron binding proteins, transferrin and lactoferrin. Tests of nineteen complexes of enterochelin as potential antibacterial agents showed that only those containing either indium (In3+) or scandium (Sc3+) inhibited bacterial growth. In this study, further evidence is presented which demonstrates a competition between the Sc3+ and Fe3+ complexes. The uptake of both complexes is energy dependent and is also repressed in iron-replete cells. The Sc3+ complex accumulates within the cells at 20% of the rate of the Fe3+ complex. The main components of the ferric enterochelin transport system are required for the transport of the Sc3+ complex although some Sc3+ appears to enter the cell by another route. The accumulation, within the cell, of 14C-labelled enterochelin complexes depends on the growth medium. The relationship of the size of the metal ion to the biological activity of the complex is discussed and possible mechanisms of action of the Sc3+ complex are considered.
