Biomedical subjects

E Smiley

Publications and source records attributed to E Smiley.

14 recordsLinked to original sources

PubMed2002

Specialist health services for people with intellectual disability in Scotland.

BACKGROUND: People with intellectual disability (ID) are known to have a high prevalence of health needs, and to require access to specialist health services in addition to primary care and generic secondary care health services. However, there is no national database of each locality's specialist health service provision. Such a record would highlight variation in provision and enable benchmarking. METHOD: A 15-item questionnaire was developed which included questions on ID health services and staffing levels. This was sent to the chief executive of each of the 15 identified National Health Service primary care trusts/health boards which provide ID services in Scotland. The same questionnaire was also sent to the lead clinician/clinical director of each service. The results were converted to per 100 000 population per trust and presented in cumulative frequency tables to allow benchmarking. RESULTS: A response rate of 100% was achieved. The results show a wide range in the type of services provided by each locality in Scotland. Only three services (21%) have completed the process of resettlement. There was a wide-ranging variability in the number of beds/day places and professionals employed per 100 000 population per trust. CONCLUSIONS: There is widespread diversity in the service provision between different parts of Scotland. Geographical distances and responsibilities for service provision to remote and rural communities did not appear to account for these differences.

Adolescent↗

PubMed1999

Engineered smooth muscle tissues: regulating cell phenotype with the scaffold.

Culturing cells on three-dimensional, biodegradable scaffolds may create tissues suitable either for reconstructive surgery applications or as novel in vitro model systems. In this study, we have tested the hypothesis that the phenotype of smooth muscle cells (SMCs) in three-dimensional, engineered tissues is regulated by the chemistry of the scaffold material. Specifically, we have directly compared cell growth and patterns of extracellular matrix (ECM) (e.g. , elastin and collagen) gene expression on two types of synthetic polymer scaffolds and type I collagen scaffolds. The growth rates of SMCs on the synthetic polymer scaffolds were significantly higher than on type I collagen sponges. The rate of elastin production by SMCs on polyglycolic acid (PGA) scaffolds was 3.5 +/- 1.1-fold higher than that on type I collagen sponges on Day 11 of culture. In contrast, the collagen production rate on type I collagen sponges was 3.3 +/- 1.1-fold higher than that on PGA scaffolds. This scaffold-dependent switching between elastin and collagen gene expression was confirmed by Northern blot analysis. The finding that the scaffold chemistry regulates the phenotype of SMCs independent of the scaffold physical form was confirmed by culturing SMCs on two-dimensional films of the scaffold materials. It is likely that cells adhere to these scaffolds via different ligands, as the major protein adsorbed from the serum onto synthetic polymers was vitronectin, whereas fibronectin and vitronectin were present at high density on type I collagen sponges. In summary, this study demonstrates that three-dimensional smooth muscle-like tissues can be created by culturing SMCs on three-dimensional scaffolds, and that the phenotype of the SMCs is strongly regulated by the scaffold chemistry. These engineered tissues provide novel, three-dimensional models to study cellular interaction with ECM in vitro.

Animals↗

PubMed1999

Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration.

The inability to deliver growth factors locally in a transient but sustained manner is a substantial barrier to tissue regeneration. Systems capable of localized plasmid gene delivery for prolonged times may offer lower toxicity and should be well-suited for growth factor therapeutics. We investigated the potency of plasmid gene delivery from genes physically entrapped in a polymer matrix (gene activated matrix) using bone regeneration as the endpoint in vivo. Implantation of gene activated matrices at sites of bone injury was associated with retention and expression of plasmid DNA for at least 6 weeks, and with the induction of centimeters of normal new bone in a stable, reproducible, dose- and time-dependent manner.

Animals↗

PubMed1999

DNA delivery from polymer matrices for tissue engineering.

We have proposed engineering tissues by the incorporation and sustained release of plasmids encoding tissue-inductive proteins from polymer matrices. Matrices of poly(lactide-co-glycolide) (PLG) were loaded with plasmid, which was subsequently released over a period ranging from days to a month in vitro. Sustained delivery of plasmid DNA from matrices led to the transfection of large numbers of cells. Furthermore, in vivo delivery of a plasmid encoding platelet-derived growth factor enhanced matrix deposition and blood vessel formation in the developing tissue. This contrasts with direct injection of the plasmid, which did not significantly affect tissue formation. This method of DNA delivery may find utility in tissue engineering and gene therapy applications.

Animals↗

PubMed1998

Alternative splicing of LTBP-3.

LTBPs bind the 100-kDa latent TGF-beta complex and thereby regulate TGF-beta assembly, tissue localization, and function. However, the 100-kDa complex is not always associated with LTBP, and, conversely, evidence suggests that LTBP has a distinct role in the extracellular matrix. As yet, there are no data to explain how the binding interaction between LTBP and the 100-kDa complex is regulated. This report provides the first direct evidence of alternative splicing of an LTBP gene. Two alternative splice sites in the mouse LTBP-3 gene have been identified based on in vivo and in vitro studies. Alternative splicing at one site in particular was found to disrupt a structural motif involved in the binding interaction with the 100-kDa latent TGF-beta complex. Therefore, alternative splicing may represent a molecular mechanism by which the uncomplexed form of LTBP-3 is produced, and, as a corollary, by which the 100-kDa latent TGF-beta 1 complex is produced.

Adaptor Proteins, Signal Transducing↗

PubMed1998

8-Cysteine TGF-BP structural motifs are the site of covalent binding between mouse LTBP-3, LTBP-2, and latent TGF-beta 1.

The small latent TGF-beta complex often is associated with the latent TGF-beta binding protein (LTBP). Three LTBPs (LTBP-1, -2, and -3) have been isolated to date. Previous studies have shown that LTBP-1 binds the small latent TGF-beta 1 complex through a disulfide bond between an 8-cysteine structural motif of LTBP-1 (TGF-bp repeat) and the propeptide dimer of latent TGF-beta 1 (TGF-beta 1 latency associated peptide). There is indirect evidence that LTBP-2 and LTBP-3 also bind the latent TGF-beta complex, but the nature and location of the binding interaction are unknown. We have used immunoprecipitation, SDS-PAGE, and autoradiography to characterize the association between mouse LTBP-3 and the small latent TGF-beta 1 complex. We report that the second and third TGF-bp repeats of LTBP-3 covalently bind the latent complex, and we show a similar capability for the homologous TGF-bp repeats of mouse LTBP-2. The second TGF-bp repeat of LTBP-3 is unusual in that it has 9 cysteine residues instead of 8, and our results provide the first evidence that a TGF-bp repeat with an odd number of cysteine residues can covalently bind latent TGF-beta 1. Altogether, these results have important implications for TGF-beta biosynthesis and the regulation of TGF-beta activity.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Mouse latent TGF-beta binding protein-2: molecular cloning and developmental expression.

The molecular cloning and developmental expression of mouse LTBP-2 are presented here. We established the identity of the cDNA by sequence comparison (80% identity with human LTBP-2) and by chromosome localization (mouse chromosome 12, band D, a region of conserved synteny with the human LTBP-2 gene). In contrast to LTBP-1 and LTBP-3, mouse LTBP-2 apparently is a more modular protein, with proline/glycine-rich sequences always alternating with clusters of cysteine-rich structural motifs. We found for the first time that LTBP-2 gene expression in mouse embryos was restricted to cartilage perichondrium and blood vessels, a somewhat surprising result since other LTBP genes are widely expressed in rodent tissues. Therefore, mouse LTBP-2 may play a critical role in the assembly of latent TGF-beta complexes in developing elastic tissues such as cartilage and blood vessel.

3T3 Cells↗

PubMed1997

Genes coding for mouse activin beta C and beta E are closely linked and exhibit a liver-specific expression pattern in adult tissues.

Five activin beta subunits have been isolated to date, and a comparative analysis of amino acid identity has suggested that the activin beta C, beta D and beta E subunits represent a distinct subset. Based on genomic cloning studies, we now report that the mouse activin beta C and beta E genetic loci are closely linked-i.e, the coding sequences are separated by 5.5-kbp. These genes also show similarities in structural organization as well as a unique liver-restricted pattern of expression in adult mice. Our results suggest that tandem duplication of an ancestral gene generated the mouse activin beta C and beta E genetic loci, and they provide further evidence for the postulate that the beta C-beta E subunits form a distinct subset of related activins. To our knowledge, this report is the first to demonstrate close chromosomal linkage between members of the TGF-beta superfamily as well as a liver-restricted expression pattern for a TGF-beta-like gene.

Activins↗

PubMed1996

Molecular cloning of the mouse activin beta E subunit gene.

cDNA clones encoding a novel activin beta subunit have been isolated from mouse liver cDNA library. An amino acid homology comparison among members of the TGF-beta superfamily indicates that the open reading frame codes for a new activin/inhibin beta subunit. The predicted mature region of the subunit shows > 60% identity with activin beta C and beta D and > or = 45% identity with activin beta A and beta B, but only 20-40% amino acid sequence identity with other TGF-beta superfamily members. Therefore, the novel subunit has been designated activin beta E. Alignment of the mature growth factor region of the five activin beta subunits revealed that activin beta A and beta B share 63% amino acid identity, while activin beta C, beta D, and beta E share 62% identity. Therefore, activin beta A and beta B are most related to one another, while activin beta C, beta D, and beta E may represent a subset of related sequences.

Activins↗

PubMed1996

Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.

Animals↗

PubMed1995

Isolation of a novel latent transforming growth factor-beta binding protein gene (LTBP-3).

This paper reports the molecular cloning of a novel gene in the mouse that shows structural similarities to the microfibril protein fibrillin and to the latent transforming growth factor-beta (TGF-beta) binding protein (LTBP), a component of the latent TGF-beta complex. The gene was initially isolated during a low stringency polymerase chain reaction screen of a NIH 3T3 cell cDNA library using primers that amplify a human fibrillin-1 epidermal growth factor-like repeat. Three lines of evidence suggest that the mouse gene is a third member of the LTBP gene family, which we designate LTBP-3. First, the deduced polypeptide, which consists of 15 epidermal growth factor-like repeats, 3 TGF binding protein repeats, and 2 proline- and glycine-rich sequences, shows 38.4% identity with LTBP-1 but only 27% identity with fibrillin-1. Second, the gene appears to be co-expressed in developing mouse tissues with TGF-beta. Third, immunoprecipitation studies using mouse preosteoblast MC3T3-E1 cells and a specific anti-peptide polyclonal antiserum reveal that the mouse polypeptide forms a complex with the TGF-beta 1 precursor. Finally, we note that the LTBP-3 gene was recently localized to a distinct genetic locus (Li, X., Yin, W., Perez-Jurado, L., Bonadio, J., and Francke, U. (1995) Mamm. Genome 6, 42-45). Identification of a third binding protein provides further insight into a mechanism by which latent TGF-beta complexes can be targeted to connective tissue matrices and cells.

3T3 Cells↗

PubMed1995

Primary structure and developmental expression of Fbn-1, the mouse fibrillin gene.

Previous studies have reported > 10 kilobases of human fibrillin-1 cDNA sequence, but a consensus regarding the 5' end of the transcript remains to be worked out. One approach to developing a clear consensus would be to search for regions of evolutionary conservation in transcripts from a related species such as mouse. As reported here, the mouse fibrillin-1 transcript encodes a highly conserved polypeptide of 2,871 amino acids. The upstream sequence that flanks the ATG is considerably less well conserved, however. Indeed, the ATG codon (which occurs in the context of a Kozak consensus sequence and is located just upstream of a consensus signal peptide) signals the point where human and mouse fibrillin-1 sequences cease to be nearly identical. Together, these results are consistent with previous efforts by Pereira et al. (Pereira, L., D'Alessio, M., Ramirez, F., Lynch, J. R., Sykes, B., Pangilinan, T., and Bonadio, J. (1993) Human Mol. Genet. 2, 961-968) to identify the human fibrillin-1 translational start site. Sequences immediately upstream of the ATG are GC-rich and devoid of TATA and CCAAT boxes, which suggests that the mouse fibrillin-1 gene will be broadly expressed. A survey of expression in mouse embryo tissues is consistent with this hypothesis and suggests two novel functions for fibrillin-associated microfibrils in non-elastic connective tissues.

Amino Acid Sequence↗

PubMed1989

RNA sequence analysis of a perinatal lethal osteogenesis imperfecta mutation.

The perinatal lethal form of osteogenesis imperfecta often results from mutations which disrupt stable assembly, delay secretion, and cause excessive posttranslational modification of type I procollagen molecules. One such mutation was efficiently characterized by an indirect method of RNA sequence analysis. The mutation initially was localized in procollagen by mapping the distribution of abnormal posttranslational modification within the triple helical domain of mutant molecules. Total RNA was isolated from osteogenesis imperfecta cells in culture, cDNA was synthesized using alpha 1(I) and alpha 2(I) specific primers, and fragments of cDNA suspected to harbor the mutation were amplified by the polymerase chain reaction technique and then cloned in M13 vectors. Sequence analysis of the amplified cDNA revealed a new, heterozygous Gly----Val substitution at residue 256 of the triple helical domain of alpha 1(I) chains produced by the perinatal lethal osteogenesis imperfecta cells. The nature and location of the mutation were confirmed by sequence analysis of amplified genomic DNA. A Gly----Val substitution has not previously been associated with the lethal form of osteogenesis imperfecta, and this mutation has the most amino-terminal location within the alpha 1(I) chain triple helical domain reported to date.

Alleles↗

PubMedDate not supplied

Low molecular weight disulfide cross-linking peptides as nonviral gene delivery carriers.

Cross-linking peptides have been developed by inserting multiple Cys residues into a 20 amino acid condensing peptide that polymerizes through disulfide bond formation when bound to DNA resulting in small, highly stable DNA condensates that mediate efficient in vitro gene transfer [McKenzie et al. (2000) J. Biol. Chem. 275, 9970-9977]. In the present study, a minimal peptide of four Lys and two terminal Cys residues was found to substitute for Cys-Trp-(Lys)(17)-Cys, resulting in DNA condensates with similar particle size and gene expression in HepG2 cells. Substitution of His for Lys residues resulted in an optimal peptide of Cys-His-(Lys)(6)-His-Cys that, in addition to the attributes described above, also provided buffering capacity to enhance in vitro gene expression in the absence of chloroquine. The reported structure-activity relationships systematically explore peptides with combinations of Lys, Cys, and His residues resulting in low molecular weight peptides with improved gene transfer properties.

Amino Acid Sequence↗