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Biomedical subjects

F Ramirez

Publications and source records attributed to F Ramirez.

At least 37 records · Page 2Linked to original sources
PubMed1991

Patient with multiple congenital anomalies and decreased production and processing of procollagen in cultured fibroblasts.

We report on a patient with hip and elbow dislocations, joint hyperextensibility, peculiar facial appearance, torticollis, cryptorchidism, unilateral hexadactyly, and other minor anomalies. Cultured cells from this patient produce less type I procollagen and have a slower rate of processing of type I procollagen to collagen in the culture medium. We think that the pattern of clinical anomalies constitutes a previously unreported syndrome with type I procollagen defect as a manifestation of the syndrome.

Abnormalities, Multiple↗
PubMed1991

Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes.

Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by skeletal, cardiovascular and ocular abnormalities. The incidence of the disease is about 1 in 20,000, with life expectancy severely reduced because of cardiovascular complications. As the underlying defect is unknown, MFS diagnosis is based solely on clinical criteria. Certain phenotypic features of MFS are also shared by other conditions, which may be genetically distinct entities although part of a clinical continuum. Immunohistochemical studies have implicated fibrillin, a major component of elastin-associated microfibrils, in MFS aetiology. Genetic linkage analysis with random probes has independently localized the MFS locus to chromosome 15. Here we report that these two experimental approaches converge with the cloning and mapping of the fibrillin gene to chromosome 15q15-21, and with the establishment of linkage to MFS. We also isolated a second fibrillin gene and mapped it to chromosome 5q23-31. We linked this novel gene to a condition, congenital contractural arachnodactyly, that shares some of the features of MFS. Thus, the cosegregation of two related genes with two related syndromes implies that fibrillin mutations are likely to be responsible for different MFS phenotypes.

Amino Acid Sequence↗
PubMed1991

G to T transversion at position +5 of a splice donor site causes skipping of the preceding exon in the type III procollagen transcripts of a patient with Ehlers-Danlos syndrome type IV.

We identified a splicing mutation in a patient with Ehlers-Danlos syndrome type IV, a heritable connective tissue disorder associated with dysfunctions of type III collagen. The mutation was first localized in the patient's type III procollagen mRNA by amplifying the reverse transcribed product in several overlapping fragments using the polymerase chain reaction. Amplified products spanning exon 24-26 sequences displayed two distinct fragments, one of normal size and the other lacking the 99 base pairs of exon 25. Sequencing of amplified genomic products identified a G to T transversion at position +5 of the splice donor site of intron 25 in one of the patient's procollagen III genes. Expression of allelic minigene constructs correlated the T for G substitution with skipping of exon 25 sequences. Like previously characterized splicing mutations in other collagen genes, lowering the temperature at which the patient's fibroblasts were incubated nearly abolished exon skipping. As a part of this study, we also identified a highly polymorphic, intronic DNA sequence whose different allelic forms can be detected easily by the polymerase chain reaction technique.

Alleles↗
PubMed1991

Multiexon deletion in the procollagen III gene is associated with mild Ehlers-Danlos syndrome type IV.

We have characterized a deletion of approximately 9 kilobases which spans from intron 33 to exon 48 of one pro-alpha 1 (III) collagen allele in a patient with Ehlers-Danlos syndrome type IV. The mutation results in the production of an in-frame species of mRNA which lacks the sequences corresponding to residues 595-1,008 of the triple-helical domain. Thus, half of the pro-alpha 1 (III) chains synthesized by the patient's fibroblasts are nearly 30% shorter than normal. The procollagen III molecules composed of either three normal length or three shortened chains are thermally stable and efficiently secreted. In contrast, the procollagen III molecules that contain one or two shortened chains are unstable and are not secreted. Failure to secrete unstable molecules and a residual functional role of the shortened but stable homotrimers may explain the somewhat milder phenotype of this individual compared with that of another Ehlers-Danlos type IV patient bearing a deletion of similar size in the amino-terminal portion of the alpha 1 (III) collagen chain.

Chromosome Deletion↗
PubMed1991

Phospholipase A2 at the bilayer interface.

Interfacial catalysis is a necessary consequence for all enzymes that act on amphipathic substrates with a strong tendency to form aggregates in aqueous dispersions. In such cases the catalytic event occurs at the interface of the aggregated substrate, the overall turnover at the interface is processive, and it is influenced the molecular organization and dynamics of the interface. Such enzymes can access the substrate only at the interface because the concentration of solitary monomers of the substrate in the aqueous phase is very low. Moreover, the microinterface between the bound enzyme and the organized substrate not only facilitates formation of the enzyme-substrate complex, but a longer residence time of the enzyme at the substrate interface also promotes high catalytic processivity. Binding of the enzyme to the substrate interface as an additional step in the overall catalytic turnover permits adaptation of the Michaelis-Menten formalism as a basis to account for the kinetics of interfacial catalysis. As shown for the action of phospholipase A2 on bilayer vesicles, binding equilibrium has two extreme kinetic consequences. During catalysis in the scooting mode the enzyme does not leave the surface of the vesicle to which it is bound. On the other hand, in the hopping mode the absorption and desorption steps are a part of the catalytic turnover. In this minireview we elaborate on the factors that control binding of pig pancreatic phospholipase A2 to the bilayer interface. Binding of PLA2 to the interface occurs through ionic interactions and is further promoted by hydrophobic interactions which probably occur along a face of the enzyme, with a hydrophobic collar and a ring of cationic residues, through which the catalytic site is accessible to substrate molecules in the bilayer. An enzyme molecule binds to the surface occupied by about 35 lipid molecules with an apparent dissociation constant of less than 0.1 pM for the enzyme on anionic vesicles compared to 10 mM on zwitterionic vesicles. Results at hand also show that aggregation or acylation of the protein is not required for the high affinity binding or catalytic interaction at the interface.

Animals↗
PubMed1991

Repeating developmental expression of G-Hox 7, a novel homeobox-containing gene in the chicken.

Here we describe the isolation and characterization of a new chicken homeobox-containing gene, G-Hox 7, which is related to Drosophila msh. The deduced amino acid sequence of the cDNA shows greater than 96% homology to the homeo domain of other vertebrate msh-like genes. As for other species, the amino and carboxy termini of the protein are, however, greatly divergent when compared phylogenetically. In situ hybridization studies revealed the early and wide-spread expression of G-Hox 7 during chick development. This includes its expression in the primitive streak and extraembryonic cells undergoing epiboly, and its expression along the neural axis, including the forebrain. Expression was also observed in the neural crest, neural crest-derived facial and branchial structures, the otocyst, limb, and heart valves. This widespread and recurrent expression of the transcript suggests that the gene may play an essential role at multiple sites during the initiation of new developmental pathways.

Amino Acid Sequence↗
PubMed1991

Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts.

The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.

Amino Acid Sequence↗
PubMed1991

Progressively restricted expression of a new homeobox-containing gene during Xenopus laevis embryogenesis.

We have isolated cDNAs encoding a novel Xenopus homeodomain-containing protein homologous to the mouse Hox-7.1 and the Drosophila muscle segment homebox (msh). Northern blot and RNAase protection experiments established that transcripts of the frog gene, termed Xhox-7.1, first appear at about the beginning of gastrulation. After a rapid increase, mRNA levels plateau between the neurula and middle-tailbud stages, and decrease steadily thereafter. In situ hybridization localized the Xhox-7.1 message to the dorsal mesodermal mantle of gastrula stage embryos. Comparison of the hybridization patterns of progressively more anterior cross-section of tailbud stage embryos localized the signal to the dorsal neural tube and neural crest, to specific regions of the lateral plate mesoderm, and to the cardiogenic region. By the tadpole stage, the Xhox-7.1 message appears only at specific sites in the central nervous system, such as in the dorsal hindbrain. Thus, during embryonic development levels of Xhox-7.1 expression decrease as the transcript becomes more progressively localized. Finally, evidence is presented of a distinct msh-like transcript (provisionally termed Xhox-7.1') which begins to accumulate at early-gastrula stage, as well.

Amino Acid Sequence↗
PubMed1991

Modifications in the organization and expression of collagen genes associated with skeletal disorders.

Fibril-forming collagens represent an evolutionary related group of structurally similar molecules within the larger family of collagen proteins. Characterization of naturally occurring mutations has provided a model whereby clinically distinct phenotypes are predicted on the basis of how specific mutations alter normal fibrillogenesis. This model, originally derived from studies of type I collagen defects in osteogenesis imperfecta and Ehlers Danlos syndrome type VII, has been modified and extended by recent correlations of type II collagen defects with several chondrodysplasias and of type III collagen defects with Ehlers Danlos syndrome type IV. From analysis of the skeletal dysplasias, the pathogenic role of fibrillar collagen defects in more common clinical entities has been suggested and awaits rigorous proof. Although informative, these collective studies have revealed important exceptions to predictions of the original pathobiochemical paradigm, and, thus, they have initiated a more rigorous reconsideration of the deductive model. As an alternative, investigations are currently underway to generate transgenic mouse models of human collagenopathies. This task will not only clarify the complexity of collagen pathophysiology, but it will also permit the development of therapeutic strategies.

Animals↗
PubMed1991

Characterization of a COL1A1 splicing defect in a case of Ehlers-Danlos syndrome type VII: further evidence of molecular homogeneity.

A child affected by the type VII form of Ehlers-Danlos syndrome (EDS VII) was shown to have a heterozygous structural defect in the amino-terminus of pro-alpha 1(I) collagen. As a result, type I procollagen trimers containing defective subunits are not converted to mature collagen molecules. To identify the cause of the protein abnormality, specifically primed cDNAs and genomic DNA were PCR amplified and sequenced. This analysis disclosed that the protein structural defect is caused by a single base substitution (A for G) at position -1 of the splice donor site of intron 6 of the pro-alpha 1(I) collagen gene (COL1A1). The affected allele produces (a) transcripts lacking exon 6 sequences and (b), in lesser amount, normally spliced transcripts. Furthermore, the rate of exon 6 skipping is temperature dependent, for it appears to decrease substantially when the patient's fibroblasts are incubated at 31 degrees C. These findings are similar to those we previously reported for other unrelated EDS VII cases and, therefore, reemphasize the molecular homogeneity of this rare connective tissue disorder.

Adult↗
PubMed1991

Characterization of a large deletion associated with a polymorphic block of repeated dinucleotides in the type III procollagen gene (COL3A1) of a patient with Ehlers-Danlos syndrome type IV.

Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant condition characterized by extreme fragility of skin, blood vessels, intestine, gravid uterus, and lungs. The phenotype is accounted for by mutations affecting the integrity and/or synthesis of the precursor procollagen molecules of type III collagen. In this article, we report the elucidation of the molecular defect in an EDS IV patient whose type III collagen was previously found to be structurally abnormal. We utilized PCR in a two-step process involving first the localization of the mutation in the mRNA and then the characterization of the defect in the gene. The results established the patient's heterozygosity for a genomic deletion of about 7.5 kb which eliminates 1,026 nucleotides of coding sequences in the message. The mutation arose as a result of an exon-to-intron recombination. The deleted segment extends from the 13th nucleotide of exon 9 to within a DNA sequence of intron 24, which is composed of a series of dinucleotide repeats. Using PCR, we tested the polymorphic nature of this DNA element on several unrelated individuals. Analysis of amplified genomic products of 45 chromosomes recognized at least four distinct allelic forms that display frequencies ranging from 5% to 61%. Mendelian segregation of three of the four alleles was established by the same method in a 3-generation family.

Alleles↗
PubMed1990

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV.

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.

Adenosine Triphosphatases↗
PubMed1990

Structural and functional characterization of a splicing mutation in the pro-alpha 2(I) collagen gene of an Ehlers-Danlos type VII patient.

A splicing mutation in the pro-alpha 2(I) collagen gene of a patient with Ehlers-Danlos syndrome type VII has been characterized. Protein microsequencing of peptides prepared from the patient's dermal collagen identified an interstitial deletion of 18 residues. The deleted segment corresponds to the amino-terminal telopeptide junction domain encoded by the sixth exon of the pro-alpha 2(I) collagen gene. Sequencing of specifically primed cDNA clones confirmed the presence of two distinct populations of pro-alpha 2(I) mRNAs, a normal one and another which lacks the sequences of exon 6. Limited sequencing of genomic clones showed that one of the pro-alpha 2(I) alleles displays a conservative change in the seventh codon of exon 6 (GAC for GAT), and a base substitution at position +1 of intron 6 (A for G). Since the normal transcript contains the GAT codon, the intronic change was associated with the allele that gives rise to the shortened pro-alpha 2(I) collagen mRNA. The two allelic fragments were subcloned into an expression vector and the pattern of splice-site selection for exons 5-8 was assessed for each of the constructs after transfection into COS cells. This documented skipping of exon 6 sequences only in transcripts of the minigene construct that harbors the G to A transition. Expression of allelic cross-constructs confirmed that the single-base substitution at position +1 of intron 6 is the mutation responsible for the abnormal joining of exons 5 and 7 sequences in the patient's shortened pro-alpha 2(I)mRNA.

Alleles↗
PubMed1990

Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen genes.

The 3500-base pair region located immediately upstream of the transcriptional start site of the human pro-alpha 2(I) collagen gene contains all the sequences necessary for cell-specific transcription. In transient expression assays, the pro-alpha 2(I) collagen promoter directed the production of high levels of bacterial chloramphenicol acetyltransferase in collagen-producing human fetal fibroblasts. Enzyme activity, on the other hand, was nearly undetectable in extracts from collagen-nonproducing immortalized lymphoblasts. Deletion experiments narrowed the active segment of the human promoter to a phylogenetically conserved sequence comprised between nucleotides-376 and -108, relative to the initiation site of transcription. In similar analyses, the pro-alpha 1(I) collagen gene failed to direct cell-specific transcription. As part of this study, the controversial issue surrounding the putative enhancer element in the first intron of the human pro-alpha 1(I) collagen gene also has been reconsidered. Accordingly, we now propose a more restricted definition of this cis-acting DNA element since its action is exerted in an orientation-preferred manner and with a strong specificity for its own promoter. Moreover, stimulation does not appear to be tissue-specific. Finally, evidence is presented supporting the notion that although structurally different and distinctly arranged, the regulatory sequences of the type I collagen genes may bind similar trans-acting factors.

Cell Line↗
PubMed1990

Cloning of a fibrillar collagen gene expressed in the mesenchymal cells of the developing sea urchin embryo.

We have cloned and characterized several overlapping cDNAs that specify a large portion of a Paracentrotus lividus fibrillar collagen molecule. Our conclusions are based on sequencing data, which showed that the clones code for a 786-amino acid collagenous domain composed of an uninterrupted series of Gly-X-Y repeats and for a 265-amino acid carboxyl-terminal globular extension. The latter domain exhibits features highly reminiscent of those of the vertebrate counterparts, notably a putative carboxyl-peptidase cleavage site, a series of similarly arranged cysteinyl residues, and an N-linked glycosylation attachment site. In situ and Northern blot hybridizations have established the size, time of appearance, and tissue localization of the collagen mRNA during sea urchin development. The collagen transcript, 9 kilobases in length, is first detected in the primary and, more predominantly, in the secondary mesenchyme cells of late gastrulae where it progressively accumulates thereafter. This and other work (D'Alessio, M., Ramirez, F., Suzuki, H.R., Solursh, M., and Gambino, R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9303-9307) provide evidence of a genetic heterogeneity of fibrillar collagens in the sea urchin embryo and suggest that the two genes are activated in the same cell lineages at distinct developmental stages.

Amino Acid Sequence↗
PubMed1990

Pro-alpha 1(XI) collagen. Structure of the amino-terminal propeptide and expression of the gene in tumor cell lines.

We have determined the nucleotide sequence of several overlapping cDNA clones encoding the amino-terminal portion of human alpha 1(XI) procollagen. These experiments have revealed that this domain of the pro-alpha(XI) chain displays structural features common to other fibrillar procollagen molecules, such as a putative amino-terminal proteinase cleavage site and an interrupted collagenous segment. In the latter, structural similarities were noted when alpha 1(XI) was compared with alpha 1(II) and alpha 2(V) procollagens. Overall, however, the amino-terminal region of pro-alpha 1(XI) differs greatly in composition and size from that of other fibrillar chains. Nearly three-fourths of this domain is in fact composed of a 383-amino acid globular region in which a 3-cysteine cluster signals the transition to a long and highly acidic carboxyl-terminal segment. Finally, the unrestricted expression of this cartilage-specific collagen gene has been confirmed by the finding of high levels of pro-alpha 1(XI) mRNA in two human rhabdomyosarcoma cell lines.

Amino Acid Sequence↗
PubMed1990

Complex and diversified regulatory programs control the expression of vertebrate collagen genes.

The collagens represent a family of structurally related but genetically distinct proteins whose function is essential to maintaining the integrity of vertebrate organs. In addition to their supportive roles, collagens influence a variety of developmental programs and physiological processes. Transcription of collagen genes is controlled by a series of complex interactions between cis-acting regulatory elements and trans-acting nuclear factors that have positive or negative effects on gene expression. Collagen synthesis relies on the timely utilization of diversified regulatory programs that employ tissue and cell-type specific promoters and enhancers. Some of these programs lead to the production of structurally variant chains in different tissues, while others shut down synthesis of a specific collagen type during cell differentiation. Still others control collagen expression in distinct cell lineages. The number, complexity, and variety of the mechanisms leading to the diversified expression of the collagen genes illustrate the unique contribution of this family of proteins to multicellular organogenesis.

Animals↗