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Biomedical subjects

B A Amendt

Publications and source records attributed to B A Amendt.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a dominant negative homeodomain mutation in Rieger syndrome.

Mutations in the PITX2 bicoid-like homeobox gene cause Rieger syndrome. Rieger syndrome is an autosomal-dominant human disorder characterized by glaucoma as well as dental hypoplasia, mild craniofacial dysmorphism, and umbilical stump abnormalities. PITX2 has also been implicated in the development of multiple organs and left-right asymmetry in the body plan. The PITX2 homeodomain has a lysine at position 50, which has been shown to impart the bicoid-type (TAATCC) DNA binding specificity to other homeodomain proteins. A mutation (K88E), found in a Rieger syndrome patient, changes this lysine to glutamic acid. We were intrigued by the relatively pronounced phenotypic consequences of this K88E mutation. In the initial analyses, the mutant protein appeared to simply be inactive, with essentially no DNA binding and transactivation activities and, unlike the wild type protein, with an inability to synergize with another transcription factor, Pit-1. However, when the K88E DNA was cotransfected with wild type PITX2, analogous to the patient genotype, the K88E mutant suppressed the synergism of wild type PITX2 with Pit-1. In contrast, a different PITX2 homeodomain mutant, T68P, which is also defective in DNA binding, transactivation, and Pit-1 synergism activities, did not suppress the wild type synergism with Pit-1. These results describe the first dominant negative missense mutation in a homeodomain and support a model that may partially explain the phenotypic variation within Rieger syndrome.

Abnormalities, Multiple↗
PubMed2001

PITX2 regulates procollagen lysyl hydroxylase (PLOD) gene expression: implications for the pathology of Rieger syndrome.

The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.

Abnormalities, Multiple↗
PubMed2001

Antagonistic regulation of Dlx2 expression by PITX2 and Msx2: implications for tooth development.

The transcriptional mechanisms underlying tooth development are only beginning to be understood. Pitx2, a bicoid-like homeodomain transcription factor, is the first transcriptional marker observed during tooth development. Because Pitx2, Msx2, and Dlx2 are expressed in the dental epithelium, we examined the transcriptional activity of PITX2 in concert with Msx2 and the Dlx2 promoter. PITX2 activated while Msx2 unexpectedly repressed transcription of a TK-Bicoid luciferase reporter in a tooth epithelial cell line (LS-8) and CHO cell line. Surprisingly, Msx2 binds to the bicoid element (5'-TAATCC-3') with a high specificity and competes with PITX2 for binding to this element. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 45-fold in CHO cells. However, it is only modestly activated in the LS-8 tooth epithelial cell line that endogenously expresses Msx2 and Pitx2. RT-PCR and Western blot assays reveal that two Pitx2 isoforms are expressed in the LS-8 cells. We further demonstrate that PITX2 dimerization can occur through the C-terminus of PITX2. Msx2 represses the Dlx2 promoter in CHO cells and coexpression of both PITX2 and Msx2 resulted in transcriptional antagonism of the Dlx2 promoter. Electrophoretic mobility shift assays demonstrate that factors in the LS-8 cell line specifically interact with PITX2. Thus, Dlx2 gene transcription is regulated by antagonistic effects between PITX2, Msx2, and factors expressed in the tooth epithelia.

Animals↗
PubMed2000

The Pitx2 protein in mouse development.

The Rieger syndrome, an autosomal dominant disorder involving ocular, dental, and umbilical defects is caused by mutations in PITX2, a Bicoid-type homeobox protein. Mouse Pitx2 mRNA is expressed in eye, tooth and umbilicus consistent with the human Riegers phenotype. Moreover, Pitx2 is involved in the Nodal/Sonic hedgehog pathway that determines left/right polarity. In this report we demonstrate a 32-kDa polypeptide on Western blots of nuclear extracts from a rat pituitary cell line, using a Pitx2 specific antibody (designated P2R10). We describe also for the first time expression of the Pitx2 protein in mouse. Pitx2 protein immunostaining was detectable during the development of the eye, tooth, umbilicus, and also in the pituitary, heart, gut, and limb. We demonstrate for the first time directly that Pitx2 is asymmetrically expressed in early heart, gut, and lung development.

Abnormalities, Multiple↗
PubMed2000

Rieger syndrome: a clinical, molecular, and biochemical analysis.

Rieger syndrome (RIEG 1; MIM 180500) is an autosomal dominant disorder of morphogenesis. It is a phenotypically heterogeneous disorder characterized by malformations of the eyes, teeth, and umbilicus. RIEG belongs to the Axenfeld-Rieger group of anomalies, which includes Axenfeld anomaly and Rieger anomaly (or Rieger eye malformation), which display ocular features only. Recently, mutations in the homeodomain transcription factor, PITX2, have been shown to be associated with Rieger syndrome. This review discusses the clinical manifestations of Rieger syndrome and how they correlate with the current molecular and biochemical studies on this human disorder.

Abnormalities, Multiple↗
PubMed1999

Transcriptional antagonism between Hmx1 and Nkx2.5 for a shared DNA-binding site.

The recently described Hmx family of homeodomain proteins is predominately expressed in discrete regions of developing sensory tissues. In this report, we have identified the preferred DNA-binding site of the murine Hmx3 homeodomain protein by the selection and amplification binding (SAAB) technique. The consensus Hmx-binding site contained the sequence 5'-CAAGTG-3', which differs from the 5'-TAAT-3' motif commonly associated with homeodomain proteins. Instead, the Hmx consensus is similar to the 5'-CAAGTG-3'-binding sites of Nkx2.1 and Nkx2.5 homeodomain proteins. Based on mutation studies, both the 5'-CAAG-3' core and the 3'-TG dinucleotide are required for high affinity binding by Hmx3 and the homologous Hmx1 protein. A critical determinant of this specificity is the glutamine at position 50 in the third helix of the Hmx homeodomain. Hmx1 binds to the 5'-CAAGTG-3' element with an apparent dissociation constant of 20 nM. Unexpectedly, the human Hmx1 protein specifically repressed transcription from a luciferase reporter gene containing 3 copies of the 5'-CAAGTG-3' sequence. In contrast, the Nkx2.5 protein transactivated this luciferase reporter. Interestingly, co-expression of Hmx1 and Nkx2.5 attenuated each others activity, suggesting that genes containing the CAAGTG element can integrate signals from these proteins. Therefore, Hmx1 and Nkx2. 5 proteins bind a unique DNA sequence and act as transcriptional antagonists.

Base Sequence↗
PubMed1999

Multifunctional role of the Pitx2 homeodomain protein C-terminal tail.

Pitx2 is a newly described bicoid-like homeodomain transcription factor that is defective in Rieger syndrome and shows a striking leftward developmental asymmetry. We have previously shown that Pitx2 (also called Ptx2 and RIEG) transactivates a reporter gene containing a bicoid enhancer and synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. In this report, we focused on the C-terminal region which is mutated in some Rieger patients and contains a highly conserved 14-amino-acid element. Deletion analysis of Pitx2 revealed that the C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2-Pit-1 interaction and Pit-1 synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Interestingly, cotransfection of an expression vector encoding the C-terminal 39 amino acids of Pitx2 specifically inhibits Pitx2 transactivation activity. In contrast, the C-terminal 39-amino-acid peptide interacts with Pitx2 to increase its DNA binding activity. These data suggest that the C-terminal tail intrinsically inhibits the Pitx2 protein and that this inhibition can be overcome by interaction with other transcription factors to allow activation during development.

Abnormalities, Multiple↗
PubMed1998

The molecular basis of Rieger syndrome. Analysis of Pitx2 homeodomain protein activities.

Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoid element in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparent KD and only about a 2-fold reduction in the Bmax. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome.

Amino Acid Sequence↗
PubMed1997

Splicing efficiency of human immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2.

We have previously demonstrated that an exon splicing silencer (ESS) is present within human immunodeficiency virus type 1 (HIV-1)tat exon 2. This 20 nucleotide (nt) RNA element acts selectively to inhibit splicing at the upstream 3'splice site (3'ss #3) flanking this exon. In this report, we have used in vitro splicing of mutated RNA substrates to determine the sequences necessary and sufficient for the activity of the ESS. The activity of the ESS within tat exon 2 maps to a 10 nt core sequence CUAGACUAGA. This core sequence was sufficient to inhibit splicing when inserted downstream from the 3'ss of the heterologous Rous sarcoma virus src gene. Mutagenesis of the interspersed purines in the polypyrimidine tract of the tat exon 2 3'ss to pyrimidines resulted in a significant increase in splicing efficiency indicating that 3'ss#3 is suboptimal. The ESS acts to inhibit splicing at the optimized 3'splice sites of both the HIV-1 tat and RSV src constructs but with a reduced efficiency compared to its effect on suboptimal 3'splice sites. The results indicate that both the ESS and a suboptimal 3'splice site act together to control splicing at the 3'splice site flanking at exon 2.

Avian Sarcoma Viruses↗
PubMed1995

The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C alpha intron.

Recently published genomic and cDNA sequences for porcine IgA suggested that the splice acceptor site in the C alpha 1-C alpha 2 intron was an AA rather than an AG dinucleotide. This possibility was tested in an in vitro HeLa cell splicing system using an RNA substrate corresponding to the genomic DNA with the putative AA splice site. Data indicated that splicing occurred at a cryptic AG site 12 nucleotides into the C alpha 2 domain rather than at the AA site. The possibility that swine B cells could use either site was tested by preparing the cDNAs from 13 different samples representing nine animals and amplifying the segment from the first C alpha 1 nucleotide to nucleotide 532 in C alpha 2 (genomic DNA numbering system). Analysis on a 6% polyacrylamide sequencing gel revealed two polynucleotide products in most samples that differed by the expected 12 nucleotides, suggesting that swine could use both splice sites. Sequence analysis confirmed that the shorter form was spliced at the downstream site and the larger form at the apparent upstream AA site. However, when the genomic DNA from an animal expressing only the longer polynucleotide was cloned and sequenced, the upstream splice acceptor site was AG not AA. Thus the data suggested that porcine IgA occurred in two allelic forms, designated IgAa and IgAb, which differ by an apparent G to A mutation in the last nucleotide of intron 1 resulting in a short-hinged (two amino acids, IgAb) variant, in which the downstream cryptic splice site is used, as well as a "normal-hinged" (six amino acids, IgAa) variant. Evidence that IgAa and IgAb are allelic was confirmed by genotypic analyses of progeny from matings of IgAa/IgAb heterozygotes. Evidence that both transcripts are functional was confirmed by showing that serum IgA levels were similar in animals homozygous for each variant.

Alleles↗
PubMed1995

Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).

Animals↗
PubMed1995

Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.

Amino Acid Sequence↗
PubMed1994

Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 (HIV-1) RNA follows a complex splicing pathway in which a single primary transcript either remains unspliced or is alternatively spliced to more than 30 different singly and multiply spliced mRNAs. We have used an in vitro splicing assay to identify cis elements within the viral genome that regulate HIV-1 RNA splicing. A novel splicing regulatory element (SRE) within the first tat coding exon has been detected. This element specifically inhibits splicing at the upstream 3' splice site flanking this tat exon. The element only functions when in the sense orientation and is position dependent when inserted downstream of a heterologous 3' splice site. In vivo, an HIV-1 SRE mutant demonstrated a decrease in unspliced viral RNA, increased levels of single- and double-spliced tat mRNA, and reduced levels of env and rev mRNAs. In addition to the negative cis-acting SRE, the flanking 5' splice site downstream of the first tat coding exon acts positively to increase splicing at the upstream 3' splice sites. These results are consistent with hypotheses of bridging interactions between cellular factors that bind to the 5' splice site and those that bind at the upstream 3' splice site.

Alternative Splicing↗
PubMed1992

Short-chain acyl-coenzyme A dehydrogenase activity, antigen, and biosynthesis are absent in the BALB/cByJ mouse.

BALB/cByJ (J) mice have short-chain acyl-CoA dehydrogenase (SCAD) deficiency and an organic aciduria similar to that of human SCAD deficiency. [9,10(n)-3H]- and [15,16(n)-3H]palmitate oxidations in J mouse fibroblasts were 96 and 35% of control, respectively, consistent with an isolated SCAD defect. Acyl-CoA dehydrogenase activities were assayed in muscle and fibroblast mitochondria from BALB/cBy controls (Y) and SCAD-deficient J mice. Medium-chain acyl-CoA dehydrogenase (MCAD) activities were comparable in both J and Y mice from all tissues. In the presence of MCAD antiserum, SCAD activities in J mice were undetectable in both tissues. Apparent Km and Vmax values in liver mitochondria suggested a somewhat increased affinity of MCAD for butyryl-CoA in J mice, as compared with MCAD from other species. Immunoblot studies using mitochondria revealed identical apparent SCAD molecular weight in liver, muscle, and fibroblasts from Y mice and no detectable SCAD antigen in J mice; MCAD antigen was detected in comparable amounts from both Y and J mice. Radiolabeling and immunoprecipitation studies in J mouse fibroblasts revealed no SCAD synthesis, but normal MCAD synthesis. These data argue against the existence of tissue-specific SCAD isoforms in the mouse and confirm that this mouse strain is a model for the human organic aciduria resulting from this beta-oxidation defect.

Acyl-CoA Dehydrogenase↗
PubMed1989

Short-chain acyl-coenzyme A dehydrogenase deficiency in mice.

A murine model for short-chain acyl-coenzyme A dehydrogenase (SCAD) deficiency has been identified and characterized in BALB/cByJ mice. These mice have undetectable SCAD activity, severe organic aciduria; excreting ethylmalonic and methylsuccinic acids and N-butyrylglycine, and develop a fatty liver upon fasting or dietary fat challenge. The mutant mice develop hypoglycemia after an 18-h fast, and have elevated urinary and muscle butyrylcarnitine concentrations. Most of these findings parallel those of human disorders associated with SCAD deficiency and other beta-oxidation defects. This mouse model presents important opportunities to investigate the biology of mammalian fatty acid metabolism and the related human diseases.

Acyl-CoA Dehydrogenase↗
PubMed1988

Long-chain acyl-coenzyme A dehydrogenase deficiency: biochemical studies in fibroblasts from three patients.

We studied fibroblasts from three patients with long-chain acyl-coenzyme A dehydrogenase (LCADH) deficiency; siblings H.C. and J.C. had milder clinical phenotypes than unrelated patient R-1. In H.C., J.C., and R-1 oxidation of [9,10(n)-3H]palmitate was 50, 48, and 28% of control, respectively, with R-1 having significantly less activity than H.C. and J.C. (p less than 0.05). Assays of mitochondrial short-chain and medium-chain acyl-coenzyme A dehydrogenases were normal in H.C. and J.C. However, mitochondrial LCADH activities in all three ranged from 17 to 21% of control. Flavin adenine dinucleotide addition increased LCADH activities in all three to 27-36% of control. In the presence of monospecific medium-chain acyl-coenzyme A dehydrogenase antiseria, LCADH activity decreased 17% in controls, and fell to less than or equal to 11% of control in J.C. and R-1. The heterogeneity observed in the [3H]palmitate oxidation studies was not explained by differences in LCADH activities under any assay condition.

Acyl-CoA Dehydrogenase, Long-Chain↗