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D J Prockop

Publications and source records attributed to D J Prockop.

At least 55 records · Page 3Linked to original sources
PubMed1999

Refinement of the chromosome 5p locus for familial calcium pyrophosphate dihydrate deposition disease.

Familial calcium pyrophosphate dihydrate deposition disease (CPPDD) is a disease of articular cartilage that is radiographically characterized by chondrocalcinosis due to the deposition of calcium-containing crystals in affected joints. We have documented the disease in an Argentinean kindred of northern Italian ancestry and in a French kindred from the Alsace region. Both families presented with a common phenotype including early age at onset and deposition of crystals of calcium pyrophosphate dihydrate in a similar pattern of affected joints. Affected family members were karyotypically normal. Linkage to the short arm of chromosome 5 was observed, consistent with a previous report of linkage of the CPPDD phenotype in a large British kindred to the 5p15 region. However, recombinants in the Argentinean kindred have enabled us to designate a region<1 cM in length between the markers D5S416 and D5S2114 as the CPPDD locus.

Calcium Pyrophosphate↗
PubMed1999

Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene.

Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.

Alternative Splicing↗
PubMed1999

Splicing mutations of 54-bp exons in the COL11A1 gene cause Marshall syndrome, but other mutations cause overlapping Marshall/Stickler phenotypes.

Stickler and Marshall syndromes are dominantly inherited chondrodysplasias characterized by midfacial hypoplasia, high myopia, and sensorineural-hearing deficit. Since the characteristics of these syndromes overlap, it has been argued whether they are distinct entities or different manifestations of a single syndrome. Several mutations causing Stickler syndrome have been found in the COL2A1 gene, and one mutation causing Stickler syndrome and one causing Marshall syndrome have been detected in the COL11A1 gene. We characterize here the genomic structure of the COL11A1 gene. Screening of patients with Stickler, Stickler-like, or Marshall syndrome pointed to 23 novel mutations. Genotypic-phenotypic comparison revealed an association between the Marshall syndrome phenotype and splicing mutations of 54-bp exons in the C-terminal region of the COL11A1 gene. Null-allele mutations in the COL2A1 gene led to a typical phenotype of Stickler syndrome. Some patients, however, presented with phenotypes of both Marshall and Stickler syndromes.

Abnormalities, Multiple↗
PubMed1999

Enhanced in situ detection of beta-glucuronidase activity in murine tissue.

We outline here a protocol for high-resolution in situ localization of beta-glucuronidase in murine tissues processed in glycol methacrylate (GMA). Murine tissues were first stained with 5-bromo-4-chloro-3-indolyl-beta-d-glucuronic acid (x-gluc), followed by histological processing in GMA. Retention of the blue indigo reaction product after overnight incubations in x-gluc allowed high-resolution localization of beta-glucuronidase activity by brightfield microscopy. When illuminated under darkfield, the x-gluc signal was enhanced, permitting detection even in cells with low-level enzyme activity. This technique offers for the first time a more sensitive enzyme histochemical method of detecting beta-glucuronidase activity in animal tissues and also the opportunity to examine expression at high magni-fication.

Animals↗
PubMed1998

Recombinant procollagen II: Deletion of D period segments identifies sequences that are required for helix stabilization and generates a temperature-sensitive N-proteinase cleavage site.

A cDNA cassette system was used to synthesize recombinant versions of procollagen II in which one of the four blocks of 234 amino acids that define a repeating D periods of the collagen triple helix were deleted. All the proteins were triple helical and all underwent a helix-to-coil transition between 25 and 42 degreesC as assayed by circular dichroism. However, the details of the melting curves varied. The procollagen lacking the D1 period unfolded 3 degreesC lower than a full-length molecule. With the procollagen lacking the D4 period, the first 25% of unfolding occurred at a lower temperature than the full-length molecule, but the rest of the structure unfolded at the same temperature. With the procollagen lacking the terminal D0.4 period, the protein unfolded 3 degreesC lower than the full-length molecule and a smaller fraction of the protein was secreted by stably transfected clones than with the other recombinant procollagens. The results confirmed previous suggestions that the collagen triple helix contains regions of varying stability and they demonstrated that the two D periods at the end of the molecule contain sequences that serve as clamps for folding and for stabilizing the triple helix. Reaction of the recombinant procollagens with procollagen N-proteinase indicated that in the procollagen lacking the sequences, the D1 period assumed an unusual temperature-sensitive conformation at 35 degreesC that allowed cleavage at an otherwise resistant Gly-Ala bond between residues 394 and 395 of the alpha1(II) chain.

Binding Sites↗
PubMed1998

Heterozygous glycine substitution in the COL11A2 gene in the original patient with the Weissenbacher-Zweymüller syndrome demonstrates its identity with heterozygous OSMED (nonocular Stickler syndrome).

The original patient with the Weissenbacher-Zweymüller syndrome was analyzed for mutations in two candidate genes expressed in cartilage (COL2A1 and COL11A2). No mutations were found in the COL2A1 gene but the COL11A2 gene contained a single-base mutation that converted a codon for an obligate glycine to a codon for glutamate at position alpha 2-955 (G955E). The results here and those published previously indicate that the Weissenbacher-Zweymüller syndrome (heterozygous OSMED), nonocular Stickler syndrome, and homozygous OSMED are all caused by mutations in the COL11A2 gene.

Abnormalities, Multiple↗
PubMed1998

Inhibition of the self-assembly of collagen I into fibrils with synthetic peptides. Demonstration that assembly is driven by specific binding sites on the monomers.

A series of experiments were carried out to test the hypothesis that the self-assembly of collagen I monomers into fibrils depends on the interactions of specific binding sites in different regions of the monomer. Six synthetic peptides were prepared with sequences found either in the collagen triple helix or in the N- or C-telopeptides of collagen I. The four peptides with sequences found in the telopeptides were found to inhibit self-assembly of collagen I in a purified in vitro system. At concentrations of 2.5 mM, peptides with sequences in the C-telopeptides of the alpha1(I) and alpha2(I) chain inhibited assembly at about 95%. The addition of the peptide with the alpha2-telopeptide sequence was effective in inhibiting assembly if added during the lag phase and early propagation phase but not later in the assembly process. Experiments with biotinylated peptides indicated that both the N- and C-telopeptides bound to a region between amino acid 776 and 822 of the alpha(I) chain. A fragment of nine amino acids with sequences in the alpha2-telopeptide was effective in inhibiting fibril assembly. Mutating two aspartates in the 9-mer peptide to serine had no effect on inhibition of fibril assembly, but mutating two tyrosine residues and one phenylalanine residue abolished the inhibitory action. Molecular modeling of the binding sites demonstrated favorable hydrophobic and electrostatic interactions between the alpha2telopeptide and residues 781-794 of the alpha(I) chain.

Amino Acid Sequence↗
PubMed1998

Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts.

Neurotransplantation has been used to explore the development of the central nervous system and for repair of diseased tissue in conditions such as Parkinson's disease. Here, we examine the effects of direct injection into rat brain of human marrow stromal cells (MSCs), a subset of cells from bone marrow that include stem-like precursors for nonhematopoietic tissues. Human MSCs isolated by their adherence to plastic were infused into the corpus striatum. Five to 72 days later, brain sections were examined for the presence of the donor cells. About 20% of the infused cells had engrafted. There was no evidence of an inflammatory response or rejection. The cells had migrated from the injection site along known pathways for migration of neural stem cells to successive layers of the brain. After infusion into the brain, the human MSCs lost their immunoreactivity to antibodies for collagen I. Initially, the human cells continued to stain with antibodies to fibronectin but the region of staining with fibronectin was significantly decreased at 30 and 72 days. The results suggest that MSCs may be useful vehicles for autotransplantation in both cell and gene therapy for a variety of diseases of the central nervous system.

Adult↗
PubMed1998

Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing.

Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for scanning PCR products for the presence of single-base and larger base mismatches in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE. With the improved conditions, CSGE detected all 76 previously identified single-base changes in a large series of PCR products from collagen genes that contain multiple exons with highly repetitive and GC-rich sequences. In a survey of 736 alleles of collagen genes, CSGE detected 223 unique single-base mismatches that were confirmed by nucleotide sequencing. CSGE has the advantage over other methods for scanning PCR products in that it is simple, requires no special preparation of PCR products, has a large capacity, and does not use radioactivity.

Base Sequence↗
PubMed1998

Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta.

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4-19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4-6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.

Animals↗
PubMed1998

Five families with arginine 519-cysteine mutation in COL2A1: evidence for three distinct founders.

Arginine519-cysteine mutation in the type II procollagen gene (COL2A1) is known to be associated with mild spondyloepiphyseal dysplasia (SED) and precocious generalized osteoarthritis (OA). Five families have now been identified with this mutation. To determine whether a common founder was responsible for the mutation in these five families, we defined the haplotype of the mutation-bearing chromosome using four restriction fragment length polymorphisms (RFLPs) and the 3'-untranslated region VNTR. Haplotype frequencies were estimated for 69 control samples. Three distinct mutation-bearing haplotypes were identified, with three families sharing a common haplotype. For three distinct haplotypes to have derived from a single founder, three independent recombination events would have had to occur. Thus the arg519 codon appears to represent a possible site of recurrent mutations in COL2A1, an uncommon phenomenon in collagen genes.

Arginine↗
PubMed1998

The collagen fibril: the almost crystalline structure.

The structure of collagen fibrils has intrigued many investigators over the years. A crystal structure has been available for some time, but the crystal structure has been difficult to reconcile with other observations about collagen fibrils such as their roundness and their growth from paraboloidal tips. Several alternative models recently have been suggested, but none of them fully account for all the data. One recent approach to solving the fibrillar structure is to define specific binding sites on the collagen monomer that direct self-assembly of monomers into fibrils.

Animals↗
PubMed1998

Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signaling.

As soon as procollagen precursors of fibrillar collagens were discovered in the early 1970s, it became apparent that connective tissues must contain proteolytic activities that cleave the N-propeptides and the C-propeptides from procollagens. Isolation and characterization of the enzymic activities, however, proved to be unexpectedly difficult. Both proteinases are large and are synthesized in several different forms with polypeptide chains ranging in size from 70 kDa to about 130 kDa. The N-proteinase has the unusual property of cleaving the N-propeptides from type I and type II procollagens if the proteins are in a native conformation, but not if the proteins are partially unfolded so that the N-telopeptides are no longer in a hair-pin configuration. The C-proteinase specifically cleaves native and denatured types I, II and III procollagens. It also specifically cleaves a precursor of lysyl oxidase and laminin 5. Both enzymes and their variants have structures that place them in a large and expanding super-family of over 200 zinc-binding metalloproteinases. The larger of two forms of the N-proteinase contains an RGD sequence for binding through integrins and properdin repeats similar to those found in thrombospondin. The shorter 70 kDa form of the C-proteinase is identical to the protein that was previously identified as bone morphogenic protein-1. Both the 70 kDa C-proteinase and two larger forms are homologous to proteins that are expressed early in development in a variety of organisms, including Drosophila, sea urchin, and fish. Therefore, the data suggest that both the N- and C-proteinases have important biological functions in addition to the roles in the processing of procollagens.

Animals↗
PubMed1998

A 1,064 bp fragment from the promoter region of the Col11a2 gene drives lacZ expression not only in cartilage but also in osteoblasts adjacent to regions undergoing both endochondral and intramembranous ossification in mouse embryos.

We isolated a 1,064 bp promoter fragment that extended from the 3'-end of the adjacent gene for retinoic X receptor-beta to beyond the most clearly defined start site of the mouse Col11a2 gene. The fragment was then joined to a beta-galactosidase gene and used to prepare transgenic mice. Three independent lines of transgenic mice were generated. The reporter beta-galactosidase gene was expressed in essentially all cartilaginous tissues in 15.5-day-old mouse embryos. In addition, the construct was expressed in osteoprogenitors within developing periosteum and in osteoblasts within mineralized bone. This pattern of expression was evident during both endochondral and intramembranous bone formation. Therefore, the results suggest that 1,064 bp promoter fragment can drive tissue-specific expression of the Col11a2 gene.

Animals↗