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Biomedical subjects

S L Woo

Publications and source records attributed to S L Woo.

At least 19 recordsLinked to original sources
PubMed1992

Structural characterization of the 5' regions of the human phenylalanine hydroxylase gene.

Human phenylalanine hydroxylase (PAH) is expressed in a liver-specific manner and catalyzes the enzymatic conversion of phenylalanine to tyrosine. Genetic deficiency of PAH results in the autosomal-recessive disorder phenylketonuria (PKU). Through the application of genomic and cDNA cloning, primer extension studies, SI mapping experiments, and PCR methodologies, the transcription initiation (CAP) site has been identified and the 5'-flanking region determined. The most upstream CAP site for the human hepatic PAH gene transcript is located 154 nucleotides upstream of the first translation codon. The genomic and cDNA sequences analyzed demonstrated that the previously reported cDNA sequence, phPAH247 [Kwok et al. (1985) Biochemistry 24, 556-561], contained a 164-nucleotide cloning artifact at its 5'-end. The 319 base pair region immediately upstream of the CAP site is characterized by the lack of a proximal TATA box and the presence of sequences similar to GC boxes, CACCC boxes, CCAAT boxes, activator protein 2 (Ap-2) sites, partial glucocorticoid response elements (GREs), and partial cyclic AMP response elements (CREs). This suggests that the human PAH gene has a TATA-less promoter regulated by multiple transcription factors.

Base Sequence↗
PubMed1992

Tissue- and development-specific expression of the human phenylalanine hydroxylase/chloramphenicol acetyltransferase fusion gene in transgenic mice.

Human phenylalanine hydroxylase (PAH) catalyzes the conversion of L-phenylalanine to L-tyrosine. Deficiency of this enzyme results in phenylketonuria, a common genetic disorder of amino acid metabolism that causes severe mental retardation. In primates, PAH is expressed specifically in the liver, while in rodents PAH activity is also present in kidney, although at a much lower level. A 9-kilobase genomic DNA fragment at the 5' end of the hPAH gene (hPAH) was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The hPAH/CAT minigene was used to generate multiple transgenic mouse lines. In all expressing lines, CAT activity was detected predominantly in the liver and at much lower levels in the kidney. By immunohistochemical staining, CAT expression was localized to hepatocytes and renal epithelial cells, both of which also express the endogenous mouse PAH enzyme. Furthermore, both the transgene and the endogenous mouse PAH were activated at about the same stage of embryonic development in the mouse liver. These results suggest that the 9-kilobase DNA fragment flanking the 5' end of the human PAH gene contains all the necessary cis-acting elements to direct tissue- and developmental-specific expression in vivo.

Animals↗
PubMed1992

Molecular characterization of two galactosemia mutations and one polymorphism: implications for structure-function analysis of human galactose-1-phosphate uridyltransferase.

We report here the molecular characterization of two galactosemia mutations, L74P and F171S, and one polymorphism, S135L, in human galactose-1-phosphate uridyltransferase (GALT). Both galactosemia mutations result in reduced enzymatic activity when reconstructed in the cDNA and overexpressed. The polymorphism, in contrast, has near normal activity. Both mutations affect evolutionarily conserved residues, suggesting that they are functionally important, while the polymorphism occurs in a nonconserved domain which is presumably not critical for enzymatic function. The F171S mutation is close to the putative active-site nucleophile. Our data further support the notion of molecular heterogeneity of galactosemia and suggest that galactosemia mutations and GALT polymorphisms may be useful tools in highlighting different functional domains in human GALT.

Base Sequence↗
PubMed1992

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

Base Sequence↗
PubMed1992

Molecular basis of phenylketonuria and related hyperphenylalaninemias: mutations and polymorphisms in the human phenylalanine hydroxylase gene.

Mutations in the human phenylalanine hydroxylase gene producing phenylketonuria or hyperphenylalaninemia have now been identified in many patients from various ethnic groups. These mutations all exhibit a high degree of association with specific restriction fragment-length polymorphism haplotypes at the PAH locus. About 50 of these mutations are single-base substitutions, including six nonsense mutations and eight splicing mutations, with the remainder being missense mutations. One splicing mutation results in a 3 amino acid in-frame insertion. Two or 3 large deletions, 2 single codon deletions, and 2 single base deletions have been found. Twelve of the missense mutations apparently result from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutation has been observed at several of these sites, producing associations with different haplotypes in different populations. About half of all missense mutations have been examined by in vitro expression analysis, and a significant correlation has been observed between residual PAH activity and disease phenotype. Since continuing advances in molecular methodologies have dramatically accelerated the rate in which new mutations are being identified and characterized, this register of mutations will be updated periodically.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Two missense mutations causing mild hyperphenylalaninemia associated with DNA haplotype 12.

The genetic defects responsible for most phenylketonuria (PKU) and hyperphenylalaninemia (HPA) cases are located in the phenylalanine hydroxylase (PAH) gene. Approximately 50-60 mutations have been reported in Caucasians and are reflected in a wide range of clinical severities. Most mutations are linked to specific haplotypes, as defined by eight polymorphic restriction sites in the PAH gene. We hypothesized that there is at least one mild mutation linked to haplotype 12 in the Swedish PKU/HPA population, since 7 of 8 patients carrying haplotype 12 had mild HPA. Sequence analysis revealed a C-to-G transversion at the second base of codon 322, resulting in a substitution of glycine for alanine, in four mutant haplotype 12 genes, and a G-to-A transition at the second base of codon 408, resulting in a substitution of glutamine for arginine, in another three mutant haplotype 12 genes. These mutations segregated with mutant haplotype 12 alleles in nuclear families but were not present on normal or other mutant alleles. Both mutations were tested in a eukaryotic expression system in which enzyme activities of different mutant PAH enzymes reflect the relative severities of the mutations, although these in vitro activities cannot be translated directly into in vivo hepatic activities. The A322G mutant PAH had about 75% and the R408Q mutant PAH about 55% of the wild-type PAH enzyme activity. These in vitro activities are the highest reported for mutant PAH enzymes produced in the same expression system.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

A defective splice site at the phenylalanine hydroxylase gene in phenylketonuria and benign hyperphenylalaninemia among Palestinian Arabs.

Phenylketonuria (PKU) and benign hyperphenylalaninemia (HPA) result from different combinations of mutations at the locus for phenylalanine hydroxylase (PAH). While some of these mutations show widespread ethnic distribution, others are unique to specific communities. We report here the first point mutation common among Palestinian Arabs. The mutation (IVS2nt1) involves a dinucleotide substitution (Gg-->Aa) at the donor splice site of intron 2 of the PAH gene and abolishes a recognition site of the restriction enzyme MnlI. IVS2nt1 is associated with two PAH polymorphic haplotypes, 7 and 42. Homozygotes for this mutation are affected with severe, classical PKU. Compound heterozygotes carrying the IVS2nt1 allele and one of several other yet unknown mutations show different degrees of benign HPA.

Base Sequence↗
PubMed1992

Ultrastructural morphometry of anterior cruciate and medial collateral ligaments: an experimental study in rabbits.

This study presents morphometric analyses of collagen subfascicle area fraction and collagen fibril diameter distributions for the anterior cruciate (ACL) and medial collateral (MCL) knee ligaments from transmission electron micrographs of ligament cross sections of five mature, female New Zealand White rabbits. Statistically significant differences in subfascicular area fractions were found between the ACL and MCL (0.89 +/- 0.02, 0.97 +/- 0.01, respectively; p less than 0.001). Mean fibril diameters for the ACL and MCL were also significantly different (0.059 +/- 0.005, 0.085 +/- 0.011 microns, respectively; p less than 0.025). Fibril eccentricity (a measure of parallel alignment of collagen fibrils within the ligaments, defined as the ratio of minor to major axes of elliptical fibril outlines) was 0.89 +/- 0.03 and 0.85 +/- 0.08, respectively, for the ACL and MCL; these data were not significantly different (p greater than 0.1). The relative amount of variation in the pooled fibril diameter data due to variation between animals, ligaments, locations within ligaments, and among fibrils at individual locations are reported. The variation of fibril diameter distributions between the ACL and MCL was substantially greater than the variation between different locations within each ligament cross section as well as between different animals. The structural differences reported may help explain known differences in the biomechanical properties of the ACL and MCL.

Animals↗
PubMed1992

Healing of the medial collateral ligament following a triad injury: a biomechanical and histological study of the knee in rabbits.

The effect of a partial medial meniscectomy and anterior cruciate ligament (ACL) transection on medial collateral ligament (MCL) healing was studied in skeletally mature rabbits. Two groups of animals, group I (isolated MCL rupture) and group II (MCL rupture with ACL transection and partial medial meniscectomy), were examined. At 6 and 12 weeks postoperatively, histological examination of the healing MCL and biomechanical evaluation of the varus-valgus (V-V) knee rotation and tensile properties of the femur-MCL-tibia complex (FMTC) were performed. Group II animals experienced substantial joint degeneration by 6 weeks. Progressive osteophyte formation was observed adjacent to the MCL insertions along with proximal migration of the MCL tibial insertion between 6 and 12 weeks. Histologic examination of the healing MCL substance from both groups showed disorganized collagen, inflammation, and fibroblast proliferation that decreased over time. For group II knees, the V-V knee rotation was found to be significantly elevated (4.7 to 5.2 times the contralateral control), and did not decrease with time. In contrast, the V-V knee rotations of the group I specimens were 1.8 times greater than control immediately following injury, and approached control values by 12 weeks. Tensile testing of the FMTCs revealed that the ultimate load increased with time for both groups, but group I had significantly higher values than group II. The linear stiffness in group I was not different than that group II and did not increase with time. For the mechanical (material) properties of the healed MCL substance, the modulus of the healing tissue for group II was only 40% that of group I. The structural properties of the FMTC and the mechanical properties of the MCL substance from both groups at 6 and 12 weeks were significantly different from the contralateral controls. We further demonstrated that immediately after ACL reconstruction, the V-V rotation of group II knees could be restored to group I levels. Recent clinical studies of MCL healing following isolated complete ligament tears have suggested that nonoperative management without immobilization leads to excellent treatment outcome. However, in more severe injuries involving additional tissues, poor quality of the healed ligament tissue and articular degeneration are observed. Our results demonstrate the deleterious effects of an untreated triad injury on the healing of the MCL substance and its insertions. Examination of the MCL substance suggests that a much larger healing mass is formed following a triad injury, which partially compensates for inferior ligament mechanical properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Reconstitution of enzymatic activity in hepatocytes of phenylalanine hydroxylase-deficient mice.

Phenylketonuria (PKU) is a metabolic disorder secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). The recent creation of a mouse strain for PAH deficiency has provided an excellent model system to explore the possibility of its phenotypic correction by hepatic gene therapy. A recombinant retrovirus containing the mouse PAH cDNA under the transcriptional control of the human CMV promoter was constructed and used to transduce hepatocytes isolated from PAH-deficient mice. Viral-transduced hepatocytes produced dramatically higher levels of mouse PAH mRNA as compared to control mock-infected hepatocytes. The PAH mRNA was translated efficiently into PAH protein that is capable of converting phenylalanine to tyrosine in vitro. These results demonstrate that the PAH-deficient mouse hepatocytes can be readily reconstituted by retroviral-mediated gene transduction, which is a crucial step towards somatic gene therapy for PKU.

Animals↗
PubMed1992

Human gene therapy: present and future.

The hematopoietic system and the liver are two primary target organs for attempting somatic gene therapy of hereditary deficiencies. Several leading laboratories have recently been able to demonstrate that bone marrow cells from rodents and non-human primates can be successfully transduced with foreign genes, resulting in the functional expression of these genes in culture. The genetically reconstituted cells can subsequently be transplanted into X-irradiated recipients, and expression of the transduced genes is observed in the recipients for more than 6 months. Subsequently, gene transfer into peripheral T-lymphocytes in humans has been attempted, and the clinical trials are currently in progress. The liver is the other major organ under intensive investigation. Primary hepatocytes can be isolated from rodents, rabbits, and dogs, and successfully transduced with recombinant retroviruses. After autologous transplantation, long term survival of the engrafted cells in vivo has been observed. More recently, it has been shown that human hepatocytes can also be efficiently transduced with recombinant retroviruses. These experimental results have laid the foundation for somatic gene therapy of hereditary deficiencies in humans in the future.

Animals↗
PubMed1992

A comparative evaluation of the mechanical properties of the rabbit medial collateral and anterior cruciate ligaments.

The biomechanical properties of the medial collateral and anterior cruciate ligaments from 30 New Zealand White rabbits were measured. Because of its complex geometry, the ACL was divided into two portions (medial and lateral) to provide uniform loading. This allowed an examination of the intra-ligamentous properties. A laser micrometer system was used to measure the cross-sectional area for tensile stress and a video dimension analyzer was used to measure the strain. The mechanical properties (stress-strain curves) of the MCL and ACL were different, with the modulus (determined between 4 and 7% strain) in the MCL (1120 +/- 153 MPa) more than twice that of either portion of the ACL (516 +/- 64 and 516 +/- 69 MPa for the medial and lateral portions, respectively). This higher modulus correlated with the more uniform and dense appearance of the collagen fibrils examined with scanning electron microscopy (SEM).

Animals↗
PubMed1992

Identification of three novel missense PKU mutations among Chinese.

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.

Alleles↗
PubMed1992

Identification of three novel PKU mutations among Chinese: evidence for recombination or recurrent mutation at the PAH locus.

Three novel mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese classical phenylketonuria (PKU) patients. Two of these substitutions (W326X and Y356X) result in the generation of a premature stop codon, while the third (IVS-7nt2) alters an invariant dinucleotide splicing signal. These mutations together account for about 10% of all PKU alleles in the Chinese population. The W326X mutation is associated with PAH RFLP haplotype 4, the most common haplotype in Orientals, while the IVS-7nt2 mutation occurs once on a haplotype 7 chromosome. The Y356X mutation is associated with multiple haplotypes, possibly due to crossover, gene conversion, or recurrent mutation.

Asian People↗
PubMed1992

Characterization of two missense mutations in human galactose-1-phosphate uridyltransferase: different molecular mechanisms for galactosemia.

We report the molecular characterization of two novel galactosemia mutations that exhibit different molecular phenotypes. Both are of the missense type with low or no residual enzyme activity. The R148W mutation results in an unstable protein, although messenger RNA is still produced. In contrast, the L195P mutation produces stable but inactive immunoreactive protein. The R148W mutation alters an amino acid that is not evolutionarily conserved, while the L195P mutation affects a well-conserved residue nine amino acids down-stream from the putative active site nucleophile. These mutations provide evidence that different mechanisms can result in galactosemia: destabilizing mutations in any given area of the protein and missense mutations in conserved domains of the enzyme resulting in low or no activity. These two mutant alleles represent the fifth and sixth galactosemia mutations and confirm the hypothesis that galactosemia results from a multiplicity of mutations at the molecular level.

Amino Acid Sequence↗
PubMed1992

Molecular biology and genetics of alpha 1-antitrypsin deficiency.

The use of advanced recombinant DNA technology has provided an improved understanding of the human AAT deficiency phenotype by providing the amino acid sequence of several variant proteins and by allowing for the production of various cell and animal models to study the molecular and biochemical components of the retention, degradation, and accumulation of these variants in the hepatic ER. Human AAT deficiency will continue to serve as an excellent model for enhancing our current understanding of mechanisms utilized in regulating protein "traffic" in the ER and in elucidating the pathophysiologic components of AAT-related liver disease.

Animals↗