Biomedical subjects

J F Bateman

Publications and source records attributed to J F Bateman.

At least 55 records · Page 3Linked to original sources

PubMed1995

A COL2A1 mutation in achondrogenesis type II results in the replacement of type II collagen by type I and III collagens in cartilage.

An autosomal dominant mutation in the COL2A1 gene was identified in a fetus with achondrogenesis type II. A transition of G2853 to A in exon 41 produced a substitution of Gly769 by Ser within the triple helical domain of the alpha 1(II) chain of type II collagen, interrupting the mandatory Gly-X-Y triplet sequence required for the normal formation of stable triple helical type II collagen molecules, resulting in the complete absence of type II collagen in the cartilage, which had a gelatinous composition. Type I and III collagens were the major species found in cartilage tissue and synthesized by cultured chondrocytes along with cartilage type XI collagen. However, cultured chondrocytes produced a trace amount of type II collagen, which was retained within the cells and not secreted. In situ hybridization of cartilage sections showed that the chondrocytes produced both type II and type I collagen mRNA. As a result, it is likely that the chondrocytes produced type II collagen molecules, which were then degraded. The close proximity of the Gly769 substitution by Ser to the mammalian collagenase cleavage site at Gly775-Leu776 may have produced an unstable domain that was highly susceptible to proteolysis. The type I and III collagens that replaced type II collagen were unable to maintain the normal structure of the hyaline cartilage but did support chondrocyte maturation, evidenced by the expression of type X collagen in the hypertrophic zone of the growth plate cartilage.

Abortion, Induced↗

PubMed1995

Matrix deposition by a calcifying human osteogenic sarcoma cell line (SAOS-2).

Procollagen and proteoglycan biosynthesis was defined in long-term culture of a human osteogenic sarcoma cell line, SAOS-2. An osteoblast phenotype was maintained by these cells up to 40 days post-confluent in the presence of ascorbic acid. Under these conditions, cells deposited around them an extensive collagenous matrix that was able to mineralize in the presence of an exogenous phosphate donor (beta-glycerophosphate). The collagenous matrix was comprised predominantly of collagen type I with significant amounts of collagen type V, and greater than 80% of the collagen in the matrix was involved in covalent crosslinkages. With increasing time in culture there was a decrease in the collagen synthetic rate, although the collagenous matrix was maintained. The proteoglycans synthesized by nonmineralizing and mineralizing cultures were purified after biosynthetic labeling with [35S]sulfate and [3H]glucosamine. Two major species were apparent: a large chondroitin sulfate proteoglycan (CSPG), and a small chondroitin sulfate proteoglycan, decorin. In nonmineralizing cultures, decorin partitioned equally between the cell layer and culture medium, whereas the large CSPG species partitioned exclusively into the cell layer-associated matrix. In the presence of extensive mineral deposition, greater than 90% of the newly synthesized proteoglycans were secreted into the medium. Northern blot hybridization indicated that SAOS-2 cells express mRNA encoding a range of bone proteins, including decorin, osteonectin, and bone sialoprotein.

Calcinosis↗

PubMed1995

Myocardial infarction resulting from coronary artery dissection in an adolescent with Ehlers-Danlos syndrome type IV due to a type III collagen mutation.

Ehlers-Danlos syndrome encompasses a group of inherited disorders of connective tissue, some of which are characterised by abnormalities of collagen metabolism. The chromosomal location, identified genes and biochemical defects, inheritance pattern, and clinical features for the various known subtypes are outlined. Prenatal diagnosis is possible for types IV, VI, VIIA1, and VIIA2. An unusual presentation of type IV Ehlers-Danlos syndrome in a 16 year old boy with an anterior myocardial infarction resulting from dissection of the left anterior descending coronary artery is reported here. A clinical diagnosis of type IV Ehlers-Danlos syndrome was made subsequently and confirmed by the reduced production, impaired secretion, and abnormally slow electrophoretic migration of type III collagen, indicating an underlying mutation in the COL3A1 gene. This patient represents the first case of type IV Ehlers-Danlos syndrome with symptomatic coronary artery dissection.

Adolescent↗

PubMed1995

Recurrent substitutions of arginine 789 by cysteine in pro-alpha 1 (II) collagen chains produce spondyloepiphyseal dysplasia congenita.

A child with typical spondyloepiphyseal dysplasia congenita had a recurrent, heterozygous substitution of arginine 789 by cysteine in the triple helical domain of alpha 1 (II) chains of type II collagen. The amino substitution was due to the transition of cytosine 2913 to thymine in exon 41 of the COL2A1 gene. The amino acid substitution involved the Y position of a Gly-X-Y triplet.

Amino Acid Sequence↗

PubMed1994

A 5' splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated pro alpha 1(I) chains with a non-collagenous insertion destabilizing the triple helix.

A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3' limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically.

Amino Acid Sequence↗

PubMed1994

Deposition and selective degradation of structurally-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro.

Collagen matrix deposition and turnover were studied in skin fibroblasts from a control and from a patient with lethal perinatal osteogenesis imperfecta (OI) identified as a Gly667 to Arg substitution in the alpha 1(I) chain. A culture system where ascorbic acid was included to stimulate collagen matrix formation over extended culture periods was used. Serial extraction of the control cell collagen matrix confirmed that a substantial mature crosslinked collagen matrix was formed in the control fibroblast cell layer. In contrast, total collagen deposition by the OI fibroblasts was poor, with the quantity of collagen deposited only about a quarter of that of the control cells. Detailed analysis of the OI fibroblast matrix revealed that the mutant collagen chains were incorporated into the collagenous matrix. These data indicate that, when grown with ascorbate in long-term culture, OI fibroblasts reproduced the abnormal matrix deposition pattern of OI tissues in vivo. The overall dramatic reduction in collagen matrix formation was not accounted for by reduced collagen production, since during the period of matrix deposition (days 8-12) the rate of production by the OI cells was only slightly less than that of the control cells. The incorporation of the newly-synthesized OI collagen into the matrix was less efficient than in control cells, reflecting the cooperative nature of matrix deposition. The fate of this mutant collagen containing the Gly to Arg charge-change was followed in the matrix by a pulse-chase experiment and two-dimensional electrophoresis. These data demonstrated that the mutant incorporated into the matrix was unstable, with the proportion of mutant declining during the chase. The deposition of the mutant monomers into a pool more accessible to proteolytic degradation indicated that the mutant and normal collagens did not copolymerize to form collagen fibers of even collagen distribution, but rather the mutant collagen was either enriched on the exposed surfaces of mixed-composition fibers, or was unable to form copolymers efficiently and polymerized into mutant-only fibrillar assemblies more prone to proteolytic attack.

Collagen↗

PubMed1993

Genomic sequence of mouse COL1A1 encoding the collagen propeptides.

The nucleotide sequences of the mouse pro alpha 1(I) gene regions coding for the N- and C-propeptides is reported. The exon-intron structure was highly homologous to human COL1A1 and the deduced amino acid sequences of the N- and C-propeptides showed 67% and 91% identity with the human sequence. This gene sequence information will allow the production of specific gene mutations by site-directed mutagenesis to study the structure and function of these important propeptide domains.

Amino Acid Sequence↗

PubMed1993

A type I collagen reporter gene construct for protein engineering studies. Functional equivalence of transfected reporter COL1A1 and endogenous gene products during biosynthesis and in vitro extracellular matrix accumulation.

A type I collagen reporter gene construct, designed to facilitate detailed analysis of the consequences of introduced structural and regulatory mutations on collagen biosynthesis and participation in the extracellular matrix, was produced by site-directed mutagenesis of the mouse COL1A1 gene. The reporter construct, pWTCI-Ile822, carried a single base change which converted the codon for amino acid 822 of the triple helix from methionine to isoleucine. This change allowed the reporter protein, [Ile822]alpha 1(I), to be distinguished from the wild-type alpha 1(I), and quantified, by its altered CNBr cleavage pattern. In mouse Mov13 cells, which synthesize no endogenous pro alpha 1(I), reporter chains associated with endogenous pro alpha 2(I), formed pepsin-stable triple helices and were secreted efficiently from the cell. The thermal stability of wild-type molecules and molecules containing the reporter [Ile822]alpha 1(I) chains was identical. The biosynthetic characteristics of wild-type and reporter chains were directly compared in stably transfected 3T6 cells. These cells did not make a distinction between reporter and endogenous alpha 1(I) chains, which were secreted from the cells at the same rate and were processed and deposited into the 3T6 cell in vitro accumulated extracellular matrix with equal efficiency. These data demonstrate that the helical sequence alteration in the reporter protein is functionally neutral and that the reporter construct, pWTCI-Ile822, is a suitable vector for the analysis of the biochemical effects of site-directed mutations in the putative COL1A1 functional domains.

Amino Acid Sequence↗

PubMed1993

Chemical cleavage method for the detection of RNA base changes: experience in the application to collagen mutations in osteogenesis imperfecta.

We discuss the definition of mutations in osteogenesis imperfecta (OI) using a chemical cleavage method for detecting mismatched bases in patient mRNA: control cDNA heteroduplexes. The method is based on the increased chemical modification of cytosines (Cs) by hydroxylamine and thymines (Ts) by osmium tetroxide when they are not paired with their complementary base. The DNA is then cleaved at the modified base with piperidine and the use of radioactively labeled DNA probes allows the position of the mismatched base to be determined by electrophoresis of the cleavage-product. The precise mutations are then determined by specific amplification and sequencing of the region containing the mismatched base. In perinatally lethal OI (OI type II) mismatches have been detected in all 17 cases studied; 12 of these have been fully characterized. In 7 of these 12 cases the mismatches were point mutations in the genes for pro alpha 1(I) or pro alpha 2(I) which resulted in glycine substitutions in the triple helical region of the protein. Sequence variation was detected in addition to the glycine substitutions in 2 cases. In 2 cases the RNA mismatch resulted from changes in the amino acid sequence of the C-propeptide domain. In the 3 remaining cases the mismatch resulted from silent nucleotide sequence variants. In the less severe forms of OI we have studied, mismatches have been detected and characterized in 8 of 12 cases. In 4 of these 8 cases the mismatch resulted from presumably neutral sequence variation and in the other 4 cases mutations have been defined.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

PubMed1993

A mouse 3T6 fibroblast cell culture model for the study of normal and protein-engineered collagen synthesis and deposition into the extracellular matrix.

Mouse 3T6 fibroblasts deposited an organized collagenous extracellular matrix during long-term culture in the presence of ascorbic acid. The matrix produced by the cells had a similar distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III and V collagens. By day 8 of culture more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction. Incorporation of NaB3H4 into reducible crosslinks and aldehydes directly demonstrated the involvement of the alpha 1 (I)CB6 and alpha 2(I)CB3.5 in crosslinks. The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization. Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation. The existence of a preformed matrix did however, increase the efficiency with which newly synthesised collagen was incorporated into the pericellular matrix. At day 0, when there was no measurable matrix present, 29% of the collagen synthesised was deposited, while by day 15, 88% of the collagen was laid down in the matrix. The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix.

Aldehydes↗

PubMed1992

Characterization of three osteogenesis imperfecta collagen alpha 1(I) glycine to serine mutations demonstrating a position-dependent gradient of phenotypic severity.

Type I collagen alpha 1(I) glycine to serine substitutions, resulting from G-to-A mutations, were defined in three cases of osteogenesis imperfecta (OI). The Gly substitutions displayed a gradient of phenotypic severity according to the location of the mutation in the collagen triple helix. The most C-terminal of these, Gly565 to Ser, led to the lethal perinatal (type II) form of OI, whereas the more N-terminal mutations, Gly415 and Gly352 to Ser, led to severe OI (type III/IV) and moderate OI (type IVB) respectively. These data support the notion that glycine substitutions towards the C-terminus of the alpha 1(I) or alpha 2(I) chains will be more clinically severe than those towards the N-terminus. This results from the more disruptive effect of the mutations at the C-terminus on helix initiation and C- and N-terminal helix directional propagation. This generalization must be modified by considering the nature of the glycine substitution and the surrounding amino acid sequence, since the helix is composed of subdomains of differing stability which will affect the ability of helix re-nucleation and propagation.

Amino Acid Sequence↗

PubMed1992

Lethal perinatal osteogenesis imperfecta due to a type I collagen alpha 2(I) Gly to Arg substitution detected by chemical cleavage of an mRNA:cDNA sequence mismatch.

A single base mismatch was detected by a chemical cleavage method in heteroduplexes formed between patient mRNA and a control collagen alpha 2(I) cDNA probe in a case of osteogenesis imperfecta type II. The region of the mRNA mismatch was amplified using the polymerase chain reaction, cloned and sequenced. A heterozygous point mutation of G to C at base pair 1,774 of the collagen alpha 2(I) mRNA resulted in the substitution of glycine with arginine at amino acid position 457 of the helix. Type I collagen of alpha 1(I)- and alpha 2(I)-chains from the patient migrated slowly on electrophoresis due to increased levels of posttranslational modification of lysine. The parents' fibroblast collagen did not contain the mRNA mismatch and the collagens showed normal electrophoretic behaviour. Two-dimensional electrophoresis of the CNBr peptides from the patient's collagen confirmed the excessive posttranslational modification of the alpha 1(I)- and alpha 2(I)-chains in the CNBr peptides N-terminal to the mutation due to disruption of the obligatory Gly-X-Y triplet repeat of the helix. The mutation led to reduced procollagen secretion and helix destabilization as evidenced by a decreased thermal stability. These data lend further support to the accumulating evidence that type I collagen alpha 2(I) glycine substitution mutations result in the same spectrum of clinical severity as those in the alpha 1(I)-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Epitope specificity of antibodies to type II collagen in rheumatoid arthritis and systemic lupus erythematosus.

Antibodies to human type II collagen were examined in the sera of 105 patients with rheumatoid arthritis (RA), 44 patients with systemic lupus erythematosus (SLE) and 11 patients who fulfilled the criteria of both diseases (RA-SLE overlap), using a solid-phase radioimmunoassay (RIA). The frequencies of antibodies to native and denatured human type II collagen were 20% and 27% in RA, 14% and 16% in SLE, and 45% and 36% in RA-SLE overlap. The specificity of the antibodies was further examined by inhibition with native and denatured type II collagen, by immunoblotting on native and denatured type II collagen, and by immunoblotting on cyanogen-bromide derived polypeptides of type II collagen. We could not identify any disease-specific patterns of reactivity. Thus, in the three disease groups the antibody response was polyclonal; there were antibody populations that reacted with native and/or denatured collagen, and epitopes could be assigned to at least three CB peptides, CB10.5, CB11 and CB8.

Antibodies↗

PubMed1992

Osteogenic capacity of collagen in repair of established periodontal defects.

Periodontal bone defects were established in four dogs, with one proximal lesion and one furcation lesion in each quadrant. These defects were treated with the implantation of collagen membranes, collagen sponge or a combination of membrane and sponge, inserted between the mucoperiosteal flaps and the bone defects. Control sites were treated in a similar surgical manner to the experimental sites, but no collagen was inserted. Substantial amounts of new bone formed in those cases treated with the collagen products, especially those treated with the membrane either with or without the sponge. The membranes limited the infiltration of small round cells, whereas in the control sites, inflammatory cells infiltrated to the bone surface. New connective tissue attachment was established in experimental situations, especially with the use of the membranes alone or in conjunction with sponge.

Animals↗

PubMed1991

Characterization of a type I collagen alpha 2(I) glycine-586 to valine substitution in osteogenesis imperfecta type IV. Detection of the mutation and prenatal diagnosis by a chemical cleavage method.

A chemical cleavage method for detecting mismatched bases in heteroduplexes formed between patient mRNA and control cDNA probes was employed to identify a single base mutation in a heterozygous case of osteogenesis imperfecta type IV. The parents' fibroblast mRNA did not contain the mutation. The region of the mRNA mismatch was amplified by using the polymerase chain reaction, cloned and sequenced. A point mutation of G to U at base-pair 2162 of the collagen alpha 2(I) mRNA resulted in the substitution of glycine by valine at amino acid position 586 of the helix. This substitution disrupted the critical Gly-Xaa-Yaa repeating unit of the collagen triple helix and resulted in helix destabilization, as evidenced by a decreased thermal stability. This local disturbance to helix propagation from the C-terminus to the N-terminus led to the overmodification of the collagen helix downstream towards the N-terminus. However, collagen secretion in vitro was normal, and the clinical phenotype probably resulted from the secretion into the extracellular matrix of the mutant collagen combined with a decrease in collagen production to 65% of control values. The rapid detection of the osteogenesis imperfecta mutation by using the chemical cleavage method afforded the opportunity to apply the technique to prenatal diagnosis in the next pregnancy of the mother of the osteogenesis imperfecta patient. The absence of a mismatched base in chorionic villus mRNA and control cDNA heteroduplexes indicated that the foetus did not carry the mutation, which was confirmed by the subsequent delivery of a normal baby.

Amino Acid Sequence↗

PubMed1991

Heterogeneity in dermatosparaxis is shown by contraction of collagen gels.

Dermal fibroblasts from sheep exhibiting a mild form of dermatosparaxis were able to contract reconstituted, fibrillar collagen gels at the same rate as control dermal fibroblasts, indicating a normal interaction between the cells and a collagenous matrix. An extract from dermatosparactic skin was shown, after partial purification, to have N-proteinase activity, although the level of activity was much lower than found in normal skin. These data show that dermatosparaxis is a heterogeneous disease, since in the severe forms of the disease the defect has been characterized as an absence of N-proteinase and an inability of the cells to interact with and contract collagen gels.

Animals↗

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

Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro.

Procollagen biosynthesis and matrix deposition were studied in long-term human skin fibroblast cultures exposed to ascorbic acid. Ascorbic acid specifically stimulated types I and III collagen synthesis, reaching a maximum at day 2 and maintaining a specific high rate of production until day 10 of ascorbate exposure, after which collagen production declined. The increased level of collagen synthesis after different exposure times could also be achieved by only brief treatment (10 h) of parallel scorbutic (ascorbic-acid-deficient) cultures with ascorbic acid. This brief exposure did not result in increased collagen mRNA, thus demonstrating that the ascorbate-induced increase in collagen synthesis at all stages of ascorbic acid exposure was due to post-transcriptional mechanisms, most likely a rapid increase in type 1 collagen mRNA translational efficiency. This mechanism, rather than the transcriptional activation, was the primary response and is adequate to explain the ascorbate-induced increase in collagen synthesis. These data also demonstrate that the presence of a collagenous extracellular matrix was not involved in this collagen biosynthetic regulation. During long-term exposure (18 days) to ascorbic acid, a substantial cross-linked collagenous matrix formed, following an approximately sigmoidal time course. The most rapid matrix deposition occurred during the later days of exposure when the rate of collagen synthesis was decreasing, suggesting that the presence of a pre-existing matrix is important for further collagen accumulation. Procollagen was also efficiently processed to collagen during this phase, demonstrating that efficient procollagen processing is an important regulatory event in collagen matrix deposition.

Ascorbic Acid↗