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The embryotoxic and osteolathyrogenic effects of semicarbazide.

The osteolathyrogenic agent semicarbazide was assayed for its toxicity and teratogenicity using early embryos of the frog Xenopus laevis. The 96-h LC50 is 1504.20 mg/l while the 96-h EC50 is 76.28 mg/l. Embryo length is altered prior to the onset of other effects indicated by a reduction in stage of development. The major malformation is associated with the notochord where the notochordal sheath is reduced owing to the disruption in the maturation and/or deposition of the connective tissue fibers.

Abnormalities, Drug-Induced↗

Characterization of elastin protein and mRNA from salmonid fish (Oncorhynchus kisutch).

1. Elastin was isolated from the bulbus arteriosus of a salmonid fish. Monoclonal and polyclonal antibodies, elicited against a CNBr digest of this protein, immunoprecipitated a polypeptide of Mr 43,000 from fish cell culture medium. 2. Cell-free translation of salmon poly A+ RNA produced a protein of approximately 43 kD that was immunoprecipitated with anti-elastin antibodies. The corresponding mRNA had an approximate Mr of 2 kb. 3. Despite similarities in amino acid composition, the differences in Mr between mammalian and salmon mRNA and protein suggest a divergence of fish and higher vertebrate elastins from an earlier ancestral gene.

Amino Acids↗

New mutations in the PPBG gene lead to loss of PPCA protein which affects the level of the beta-galactosidase/neuraminidase complex and the EBP-receptor.

We describe the clinical findings, and the molecular and biochemical studies in an Italian family with recurrent hydrops fetalis due to galactosialidosis (GS). GS is a rare lysosomal storage disorder caused by a deficiency of the protective protein/cathepsin A (PPCA). This protein forms a high-molecular-weight complex with the hydrolases beta-galactosidase (GLB1) and neuraminidase (NEU1). By virtue of this association these two enzymes are correctly compartmentalized in lysosomes and protected against rapid proteolytic degradation. Controversial data show that PPCA is also present in a second complex, including the Elastin Binding Protein (EBP) the EBP-receptor, which is involved in elastogenesis, and NEU1. We investigated the potential role of the PPCA in both complexes. Two new genetic lesions (c60delG and IVS2+1 G > T) that lead to a frameshift and a premature stop codon were detected in the PPCA cDNA and genomic DNA of the patient. The deleterious effect of such mutations was confirmed by the complete absence of the PPCA protein on Western blots. Thus, we examined the effect of the loss of PPCA on the two protein complexes in the patient's fibroblasts. Interestingly, a reduced amount of both GLB1 and EBP proteins was detected. These data confirm that PPCA is present in two functional complexes one with GLB1 and NEU1 in the lysosomal lumen and the other with EBP at the cell surface. The reduction in GLB1 and EBP confirms that PPCA is essential for their integrity.

Cathepsin A↗

Predictive estimation of protein linear epitopes by using the program PEOPLE.

A single small segment (sequence recognition) or domain (conformation recognition) of a protein could act as an antigen (antigenic determinant) vs an antibody. Epitopes of the first kind being a continuous segment along the sequence (linear), generally bent with a typical non-ordered structure (turns and/or loops), can be predicted from the only knowledge of the primary structure. After reviewing the different algorithms, we present PEOPLE (Predictive Estimation Of Protein Linear Epitopes) which uses combined prediction methods, taking into account the basic fundamental properties corresponding to what should be an ideal epitope: bent (secondary structure mainly beta-turns), surface accessible, hydrophilic and mobile and/or flexible. Four classes of basic biophysical parameters are considered for the determination of an antigenic index AG - secondary structure; hydrophilicity; surface accessibility; flexibility. The AG index is finally defined as a linear combination of the four class profiles. Typical applications are presented.

Animals↗

Alternate exon usage is a commonly used mechanism for increasing coding diversity within genes coding for extracellular matrix proteins.

Extracellular matrix proteins are a diverse family of secreted proteins and glycoproteins that are responsible for a variety of critical functions in different tissues. A large number of multiexon genes encode these proteins of the extracellular matrix. Over the last few years, it has become evident that the processing of the pre-mRNA from several of these genes involves alternative splicing. This review summarizes the known examples of alternative splicing in genes coding for the extracellular matrix and attempts to relate the increase in coding diversity generated by alternate exon usage to the function(s) of individual extracellular matrix proteins.

Alternative Splicing↗

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Amino Acid Sequence↗

Translation of chick aortic elastin messenger ribonucleic acid. Comparison to elastin synthesis in chick aorta organ culture.

Studies were undertaken to define the molecular size of the elastin primary gene product. Translation of chick aortic messenger ribonucleic acid (mRNA) in an mRNA-dependent reticulocyte lysate resulted in the synthesis of two major proteins of 70 000 and 73 000 molecular weights. Both proteins were shown to be soluble forms of elastin by isotope incorporation, immunoprecipitation, collagenase and cyanogen bromide sensitivity, and two-dimensional gel electrophoresis. The 70 000-dalton protein behaves similarly to authentic tropoeleastin in sodium dodecyl sulfate gel electrophoresis. There was no evidence for a high molecular weight form of soluble elastin, although procollagen chains were indirectly identified among the aortic mRNA-directed translation products. The same molecular size proteins were also seen in organ cultures of chick embryonic aortas labeled with [3H]valine. However, the 73 000-dalton protein was not extractable in a neutral salt buffer but was found only if the aortas were extracted with urea in the presence of reducing and alkylating reagents. The results from these studies suggest that elastin is first synthesized as two distinct polypeptide chains which differ slightly in size and overall charge. The possibility that these two proteins may associate posttranslationally to form a dimer prior to secretion is postulated to explain the existence of a putative proelastin molecule seen in other systems.

Animals↗

Accumulation of elafin in actinic elastosis of sun-damaged skin: elafin binds to elastin and prevents elastolytic degradation.

Elafin has a primary structure with two functional domains; a transglutaminase substrate domain at the N-terminus and a protease inhibitor domain at the C-terminus. Elafin expression has so far been reported only for epithelial tissues. Accumulation of elafin was immunohistochemically detected in the actinic elastosis of sun-damaged skin. Exposure of normal skin to UVA induced elafin expression that colocalized with elastic fibers. Incubation of synthetic transglutaminase substrate domain of elafin and elastin molecules in the presence of tissue transglutaminase in vitro resulted in the formation of a higher molecular complex on SDS-PAGE. Elafin expression was not detected in normal cultured skin fibroblasts, but was induced by UVA irradiation at both messenger RNA and protein levels. When radiolabeled insoluble elastin was incubated with recombinant full-length elafin and tissue transglutaminase, insoluble elastin became more resistant to neutrophil elastase digestion. These results indicate that (1) dermal fibroblasts potentially express elafin on UV irradiation, (2) UV-mediated elafin interacts with elastin, and (3) the elafin-elastin complex protects elastic fibers from elastolytic degradation, leading to the accumulation of elastic fibers in the actinic elastosis of sun-damaged skin. The transglutaminase substrate moiety of elafin plays an important role in anchoring elafin at its proper sites of action during UV-induced aging processes.

Adolescent↗

Keratinocytes influence the maturation and organization of the elastin network in a skin equivalent.

Elastic fibers form a complex network that contributes to the elasticity of connective tissues. Alterations in the elastic fiber network are involved in several disease affecting organs in which compliance of the connective tissue is essential: skin, main vasculature, lung, joints, muscle, and ligament. The aim of our work was to study the deposition, maturation, and organization of elastic fiber components in a dermal equivalent model consisting of collagen-GAG-chitosan seeded with fibroblasts. The influence of keratinocytes was studied in parallel, thus constituting a skin equivalent model. These models were examined by transmission electron microscopy (TEM) and by immunohistochemistry to determine the staining patterns of fibrillin-1 and elastin proteins representative of the microfibrillar framework and of the elastic fibers, respectively. After 2 mo of fibroblast culture in the dermal equivalent, elastin was undetectable, whereas fibrillin-1 staining was weak and microfibrils were infrequently observed by TEM. In the skin equivalent, fibrillin-1 and elastin were detected by immunostaining 15 d after epidermization and TEM revealed the typical structure and organization of the elastic network in the dermis, with elastin deposition on the microfibrillar scaffold. This in vitro skin equivalent model is to our knowledge the first in which elastic fibers have been detected, thus demonstrating the influence of keratinocytes on the maturation and organization of the elastic network.

Antibodies↗

Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms.

Ascorbate contributes to several metabolic processes including efficient hydroxylation of hydroxyproline in elastin, collagen, and proteins with collagenous domains, yet hydroxyproline in elastin has no known function. Prolyl hydroxylation is essential for efficient collagen production; in contrast, ascorbate has been shown to decrease elastin accumulation in vitro and to alter morphology of elastic tissues in vivo. Ascorbate doses that maximally stimulated collagen production (10-200 microM) antagonized elastin biosynthesis in vascular smooth muscle cells and skin fibroblasts, depending on a combination of dose and exposure time. Diminished elastin production paralleled reduced elastin mRNA levels, while collagen I and III mRNAs levels increased. We compared the stability of mRNAs for elastin and collagen I with a constitutive gene after ascorbate supplementation or withdrawal. Ascorbate decreased elastin mRNA stability, while collagen I mRNA was stabilized to a much greater extent. Ascorbate withdrawal decreased collagen I mRNA stability markedly (4.9-fold), while elastin mRNA became more stable. Transcription of elastin was reduced 72% by ascorbate exposure. Differential effects of ascorbic acid on collagen I and elastin mRNA abundance result from the combined, marked stabilization of collagen mRNA, the lesser stability of elastin mRNA, and the significant repression of elastin gene transcription.

Animals↗