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The cDNA sequence of the murine proline/arginine-rich end leucine-rich repeat protein (PRELP) gene was cloned by PCR-based techniques. The gene encodes a protein of 378 amino acids, which is four amino acid residues shorter than its human counterpart. This difference resides mainly in the amino terminal region of the mature protein, which is five amino acids shorter in the mouse than the human and has a lower arginine content. The remainder of the protein, including the structure of the leucine-rich repeats, the potential sites for N-linked glycosylation, and the disulfide-bonded domains are well conserved between species. In common with humans, the murine gene possesses three exons, with the translation initiation codon residing in exon 2 and the termination codon in exon 3. Exons 1 and 2 are separated by an intron of approximately 6.7 kbp, whereas exons 2 and 3 are separated by an intron of approximately 1.7 kbp. Western blot analysis of mouse cartilage extracts indicates that PRELP exists as a glycoprotein of approximately 55 kDa, as in human cartilage. Immunohistochemical and in situ hybridization analysis reveal that PRELP is expressed in cartilage throughout both fetal development and post-natal life, in contrast to the human where expression in cartilage is not apparent prior to birth. Northern blot analysis indicates that PRELP mRNA is also expressed in the developing embryo prior to skeletogenesis. The promoter region of the mouse PRELP gene possesses no TATA box in its proximal region, in common with humans, and shows differences in the conservation of elements known to be involved in regulating expression of the human PRELP gene.

Next Section Abstract The human lumican gene was shown to possess one major transcription start site, resulting in exon 1 of the gene giving rise to the first 74 base pairs (bp) of the 5′-untranslated region. About 1.6 kilobase pairs of upstream promoter sequence were sequenced and analyzed to identify elements responsible for gene expression. No typical TATAA sequence was identified in the vacinity of the transcription start site, but an atypical TATCA sequence residing 41 bp upstream was shown to be necessary for transcription, although it was incapable of supporting transcription by itself. A GC box residing 74 bp upstream of the transcription start site also was essential for the initiation of transcription. Sp3 was identified as the transcriptional activator binding to the GC box. No additional elements that significantly modulated transcription were noted in the promoter sequence analyzed, when using human adult chondrocytes as the cell source for transfection in reporter assays. In contrast, reporter assays carried out in human fetal lung fibroblasts, where lumican expression is deplete, revealed the presence of a repressor element located between 384 and 598 bp upstream of the transcription start site. A GATA-binding site located between bp −386 and −391 was identified as being necessary for repression of transcription. The mouse lumican promoter does not possess an equivalent site, and this may explain why the lumican gene is expressed in fetal murine cartilage but not in fetal human cartilage.

The pathophysiological and biological significance of tissue inhibitor of the metalloproteinases-3 (TIMP-3) gene compared to other TIMPs was investigated in osteoarthritic (OA) human and normal bovine joint tissues. Human OA synovial fibroblasts in culture constitutively expressed TIMP mRNAs. TIMP-3, TIMP-1 and gelatinase A mRNAs were elevated in most human OA synovia over controls, while TIMP-2 expression was similar. TIMP-3 and TIMP-1 mRNAs present in bovine cartilage were inducible by serum factors. Transforming growth factor beta (TGF-beta 1) induced TIMP-3 RNA and protein in human OA and normal bovine chondrocytes. TIMP mRNAs were low (TIMP-1) or undetectable in human fetal chondrocytes but were expressed at all other ages. Thus, the two main joint tissues, synovial membranes and cartilage, express TIMP genes. Due to their matrix protecting activities, the presence of multiple TIMPs may be beneficial for normal joints, while increased TIMP-3 and TIMP-1 expression in arthritic joints may be associated with pathological remodeling.

Vitamin E is important in maintaining normal neurological structure and function. In this study, 100 children with protein-energy malnutrition (PEM) were studied and compared to a suitably age-matched control group. Posterior column deficits, cerebellar deficits, and problems with fine motor coordination were present to a significant degree in the PEM subjects. The presence of neurological signs was correlated with various parameters of vitamin E deficiency, including low serum alpha-tocopherol levels and a low tocopherol/total lipid ratio which was present in 92 per cent of subjects. There was good concordance between vitamin E levels and vitamin E to serum lipid ratio in assessing vitamin E deficiency. We conclude that vitamin E deficiency is prevalent, to a hitherto unsuspected degree, in children with PEM and that these malnourished children have significant neurological deficits attributable to low vitamin E levels. This observation is of clinical significance as the neurological deficits are potentially reversible with vitamin E supplementation.

Although current ultrasound techniques provide a linear (amniotic fluid index; AFI) or two-dimensional area index of amniotic fluid (AF), these indices have limited correlation with actual AF volume. We sought to quantify the three-dimensional volume of ultrasound-identified AF pockets, as assessed by the AFI and two-dimensional area methods. The BVI 2500 (Bladder Volume Instrument 2500; Diagnostic Ultrasound Corp., Redmond, WA) has been used to quantify the volume of residual urine in the bladder. INSTRUMENT AND METHOD: The BVI 2500 (Diagnostic Ultrasound Corp.) ultrasound uses a rotating 2-MHz transducer, computer-defined fluid interface, and computer integration of 12 cross-sectional images to calculate three-dimensional fluid volume. After providing written informed consent, 14 term pregnant patients (36-42 weeks) were evaluated using the BVI 2500 and an Ultramark 8 sector scan (Advanced Technology Laboratory, Bothell, WA). The largest vertical fluid pocket in each quadrant of the abdomen was identified with the sector scan, and vertical and horizontal measurements for AFI and two-dimensional area were recorded. Simultaneous AF volume measurements of each pocket were performed three times with the bladder volume instrument, and maximum values were used. Three-dimensional volume, two-dimensional area, and AFI values were compared by correlation analysis, with Por = .05 considered statistically significant.Among all patients, the average (+/- standard deviation) AFI was 7.6 +/- 4.1 (range 1.5-16.4) cm, and the average two-dimensional area was 30.9 +/- 21.1 (range 4.3-81.3) cm2. This corresponded to an average three-dimensional volume of 215 +/- 134 (range 23-497) cm3. Three-dimensional volume correlated highly with both AFI (r = 0.9; P.001) and two-dimensional area (r = 0.86; P.001). One AFI centimeter was equivalent to a volume of 30 cm3.There are highly significant linear correlations of three-dimensional amniotic fluid volumes with AFI and two-dimensional area. The four pockets used in AFI determination account for only 50% of total AF volume. Three-dimensional determinations may aid in clinical assessments of AF volume.

The cDNA sequence of the human chondroadherin gene was cloned using PCR-based techniques. The gene encodes a protein of 359 amino acids, of which the first 21 amino acids represent a putative signal peptide sequence and which possesses 11 leucine-rich repeats flanked by cysteine-rich regions. The cDNA possesses a 5' untranslated region of 149 bp, a coding region of 1080 bp including the stop codon, and a 3' untranslated region of 561 bp terminating in a poly(A) tail. The cDNA hybridizes with a single messenger RNA of 1.9 kb, which is present in chondrocytes at all ages. Analysis of genomic DNA revealed that the chondroadherin gene possesses two introns, both of which reside within the coding region. The first intron has a length of about 2.3 kb and separates the codons for lysine(258) and phenylalanine(259). The second intron has a length of about 0.5 kb and splits the codon for tryptophan(314). This genomic organization results in exon 1 encoding the signal peptide, the amino-terminal cysteine-rich region, and the first 9 leucine-rich repeats; exon 2 encoding the last 2 leucine-rich repeats and part of the carboxy-terminal cysteine-rich region; and exon 3 encoding the remainder of the carboxy-terminal cysteine-rich region. The gene does not possess a TATA box prior to its transcription start site. Isolation of a cosmid clone spanning the chondroadherin gene enabled its chromosome location to be established. The gene was shown to reside at chromosome 17q21.33.

The gene corresponding to a 55-kDa matrix protein previously described in adult human articular cartilage was characterized by sequencing of genomic clones. The deduced protein sequence corresponds to the recently described matrix protein PRELP. The protein was encoded by messages of 1.7, 4.6, and 6.7 kb, whose relative abundance increased as their size decreased. The message heterogeneity appears to originate from variation in the length of the 3'-untranslated region, with the smallest message being contained within the reported sequence and the larger messages having extended 3'-untranslated regions. Two introns were identified within the genomic sequence encoding the smallest message. The first intron of about 6.7 kb resides 16 nucleotides prior to the translation initiation codon, and the second intron of about 2.6 kb resides 173 nucleotides prior to the translation termination codon. The gene, which encompasses at least 16 kb of genomic DNA, was shown to reside on chromosome 1q32. Primer extension techniques were used to establish that the coding sequence commences 199 bp downstream from the major transcription start site. Analysis of the DNA sequence upstream from the transcription start site reveals the presence of numerous potential transcription factor binding sites, but no CAAT or TATA box. At the message level, gene expression was at a high level in juvenile and adult cartilage, but not in the fetus or neonate. The presence of protein in the cartilage matrix was also much lower in the neonate than in the adult. In noncartilagenous tissues appreciable message levels were observed only in the adult lung.

A human lumican cDNA sequence was derived by polymerase chain reaction techniques from RNA obtained from intestine, placenta, and articular cartilage. A contiguous sequence of 1729 bases was obtained corresponding to an observed message size of 1.8 kilobases (kb). The cDNA sequence consists of an 80-base pair (bp) 5′-untranslated region, a 1014-bp coding sequence, and a 618-bp 3′-untranslated region terminating in a 17-bp poly(A) tail. The deduced lumican protein sequence has 338 amino acids, including a putative 18-residue signal peptide. The human lumican gene was shown to be spread over about 7.5 kb of genomic DNA and to be located on chromosome 12q22. The gene consists of 3 exons separated by introns of 2.2 and 3.5 kb. The shorter 5′-intron resides 21 bases prior to the translation initiation codon, and the 3′-intron resides 152 bases prior to the translation termination codon. The lumican message is expressed at high levels in adult articular chondrocytes but at low levels in the young juvenile. This age-related trend in message level is not, however, common to all tissues in which the lumican gene is expressed. Lumican is present in the extracellular matrix of human articular cartilage at all ages, although its abundance is far greater in the adult. In the adult cartilage lumican exists predominantly in a glycoprotein form lacking keratan sulfate, whereas the juvenile form of the molecule is a proteoglycan.

The expression of six cell-surface proteoglycans (syndecan, fibroglycan, amphiglycan, glypican, betaglycan and CD44) was studied at the mRNA level. Analysis was performed by Northern blotting using total RNA preparations from freshly isolated articular chondrocytes obtained from both juveniles and adults. Similar results were obtained for both age groups. By far the most abundant message was that for amphiglycan, CD44 message was next in relative abundance, and the messages for fibroglycan, glypican and betaglycan were all expressed at low levels. Syndecan message could not be detected by this technique. This pattern of expression was different to that observed in cultured skin fibroblasts, where the messages for amphiglycan, CD44, fibroglycan and glypican were all expressed at a similar level. In contrast with the fibroblasts, where the amphiglycan message exhibits no size polymorphism, the chondrocyte amphiglycan message is present in three polymorphic forms, due to the use of alternative polyadenylation signals. When the newly isolated chondrocytes are maintained in monolayer culture for several passages, the amphiglycan message heterogeneity reverts to that characteristic of the fibroblasts. Thus human articular chondrocytes are characterized by both their high level of amphiglycan message expression and their use of alternative polyadenylation signals.

We have identified a large family with a dominantly inherited chondrodysplasia characterized by a waddling gait, short limbs, and early onset osteoarthritis. The radiographic presentation resembles pseudoachondroplasia in childhood and multiple epiphyseal dysplasia in adults. Electron microscopic examination of cartilage reveals accumulation of material within the rough endoplasmic reticulum similar to that seen in pseudoachondroplasia and the Fairbank type of multiple epiphyseal dysplasia. By linkage analysis, we have excluded the genes for aggrecan, decorin, hexabrachion (tenascin), type II procollagen, the alpha 1 chain of type XI procollagen, the alpha 1 chain of type IX procollagen, and link protein, candidate genes that encode structural components of the cartilage extracellular matrix, as the disease locus for this disorder.

Total RNA was isolated from kidneys of Sprague-Dawley rats. Oligo (dT)-primed single-stranded cDNA was obtained by the reverse transcriptase reaction from which a 285 bp cDNA probe coding for 25-hydroxyvitamin D 3 -24-hydroxylase [25(OH)D 3 -24-OHase] was generated by the polymerase chain reaction. Northern blotting performed with kidney poly (A) + RNA isolated from rats (1) fed a standard diet, (2) depleted in D 3 and hypocalcemic, and (3) fed a standard diet and injected intraperitoneally with 50,000 IU of vitamin D 3 for 5 days showed that the transcript for 24-OHase was weakly expressed in control, and highly induced in vitamin D 3 -treated animals. No transcript could be elicited in vitamin D-depleted hypocalcemic animals in which 25(OH)D 3 -1α-OHase, was maximally induced. The data show that 24-OHase is independently regulated of 1α-OHase, strongly suggestive of the enzymes being encoded by two distinct genes.

PRELP is a member of the small leucine-rich repeat proteoglycan family that is abundantly expressed in many cartilages compared to other connective tissues. To study the consequence of PRELP overexpression in tissues where it is normally expressed at low abundance, transgenic mice were generated in which the human PRELP transgene was placed under control of the CMV promoter. A connective tissue phenotype was observed in the skin, where the organization of collagen fibrils in the dermis was perturbed and the thickness of the hypodermal fat layer was diminished.

The ability to generate tissue-specific ablation of gene expression has been extremely useful in connective tissue biology, as it can potentially overcome the early embryonic lethal phenotype often associated with universal gene knockout. The value of tissue-specific knockouts can be enhanced by also allowing gene ablation to occur at specific times during development, growth or aging. In the present work a transgenic mouse has been generated in which expression of Cre recombinase is under control of both the type II collagen promoter to allow cartilage-specific expression and a doxycycline response element to permit temporal control of expression. This mouse has been crossed with the Rosa26R reporter mouse, which possesses a floxed repressor element associated with a lacZ transgene, in order to validate the functional efficacy of the conditionally expressed Cre. The results demonstrate that excision of the floxed element can be achieved specifically in cartilage at different times during embryonic and juvenile development. The conditional Cre transgenic mouse should be a valuable tool to all interested in skeletal development.

This report describes the cloning of the 3{prime}-untranslated region of the human fibromodulin cDNA and its use to map the gene. For somatic cell hybrids, the generation of the PCR product was concordant with the presence of chromosome 1 and discordant with the presence of all other chromosomes, confirming that the fibromodulin gene is located within region q32 of chromosome 1. The physical mapping of genes is a critical step in the process of identifying which genes may be responsible for various inherited disorders. Specifically, the mapping of the fibromodulin gene now provides the information necessary to evaluate its potential role in genetic disorders of connective tissues. The analysis of previously reported diseases mapped to chromosome 1 reveals two genes located in the proximity of the fibromodulin locus. These are Usher syndrome type II, a recessive disorder characterized by hearing loss and retinitis pigmentosa, and Van der Woude syndrome, a dominant condition associated with abnormalities such as cleft lip and palate and hyperdontia. The genes for both of these disorders have been projected to be localized to 1q32 of a physical map that integrates available genetic linkage and physical data. However, it seems improbable that either of these disorders, exhibiting more » restricted tissue involvement, could be linked to the fibromodulin gene, given the wide tissue distribution of the encoded proteoglycan, although it remains possible that the relative importance of the quantity and function of the proteoglycan may avry between tissues. 11 refs., 1 fig. « less

Splicing variation of the versican message and size heterogeneity of the versican core protein were analyzed in human articular cartilage and intervertebral disc. Splicing variation of the message was studied by PCR analysis to detect the presence or absence of exons 7 and 8, which encode large chondroitin sulfate attachment regions. At all ages in normal cartilage from the third trimester fetus to the mature adult, the presence of the versican isoform possessing exon 8 but not exon 7 (V1) could be readily detected. The message isoforms possessing neither exon 7 nor 8 (V3) or both exons 7 and 8 (V0) were only detectable in the fetus, and the isoform possessing only exon 7 (V2) was never detected. In osteoarthritic cartilage and in adult intervertebral disc the versican message pattern was the same as that observed in the normal adult with only the isoform possessing exon 8 being detected. Core protein heterogeneity was studied by immunoblotting following enzymic removal of the glycosaminoglycan chains from the proteoglycan, using an antibody recognizing the globular G1 region of versican. All articular cartilage extracts from the fetus to the mature adult contained multiple core protein sizes of greater than 200 kDa. The adult cartilage extracts tended to have an increased proportion of the smaller sized core proteins and osteoarthritic cartilage possessed similar core protein sizes to the normal adult. In contrast, intervertebral disc at all post-natal ages showed a greater range of size heterogeneity with a prominent component of about 50 kDa. The abundance of this component increased if the samples were treated with keratanase prior to analysis, suggesting that the G1 region of versican in disc can be substituted with keratan sulfate. The increased presence of versican in the disc relative to articular cartilage may suggest a more pronounced functional role for this proteoglycan, particularly in the nucleus pulposus.

Neurological signs including posterior column, spinocerebellar, retinal, and peripheral nerve deficits are being increasingly recognized in vitamin E deficiency states. Children suffering from protein-energy malnutrition (PEM) revealed significantly reduced serum alpha-tocopherol levels compared to age-matched normal children, the deficient subjects also exhibited the widely recognized signs of tocopherol deficiency. In this prospective therapeutic intervention study moderate PEM subjects were administered aqueous oral vitamin E supplementation for 6 weeks and compared with control PEM subjects. The parameters studied included pre- and post-therapy serum alpha-tocopherol levels, alpha-tocopherol lipid ratio, lipid profile, creatine phosphokinase levels, and electroneurophysiological studies. Vitamin E supplementation normalized serum alpha-tocopherol levels (p < 0.001), alpha-tocopherol lipid ratio (p < 0.001), reduced creatine phosphokinase levels (p < 0.01), and reduced neurological signs in PEM subjects (p < 0.001). The observed improvement in neurological dysfunction among PEM subjects is of great interest, especially in developing countries. While larger studies are recommended, the importance of vitamin E administration in PEM is being reported.

The 5´-flanking region of the human proline/arginine-rich end leucine-rich repeat protein (PRELP) gene has been characterized for both promoter and repressor activity by using a variety of reporter gene constructs and transient transfection into chondrocytes or fibroblasts. The human PRELP gene lacks a TATA box, and in its absence a Sp1-binding site residing 29 bp upstream of the transcription start site is essential for initiating gene expression. In contrast, an Ets-binding site residing 497 bp upstream of the transcription start site can lead to the repression of gene expression. The analysis of nuclear proteins by gel retardation studies with the repressor element identified a common protein, presumably an Ets family member, present in neonatal chondrocytes and skin fibroblasts that do not express the PRELP gene. The factor was not detected in nuclear protein preparations from adult chondrocytes in which the PRELP gene is expressed.

Functional recombinant human link protein has been produced using a baculovirus expression system. In addition to the intact link protein, three mutant forms have also been expressed. Each mutant bears a deletion equivalent to the protein encoded by one exon in the gene. These deletions represent the A domain, which is thought to be responsible for interaction with aggrecan, and the B or B' domains, which are associated with the interaction with hyaluronate. Such deletions split codons spanning exon boundaries, but maintain the reading frame of the protein and result in the correct amino acid being present at the splice junction. All the recombinant proteins appear as two components upon SDS/PAGe, though the abundance of the two forms does vary between preparations, as a result of variable substitution by N-linked oligosaccharides. The recombinant intact link protein was able to interact with both hyaluronate and aggrecan, showing that the baculovirus system is able to produce functional molecules. All of the recombinant mutant link proteins were also able to interact with hyaluronate, indicating that both the B and B' domains can function independently. The recombinant mutant link proteins were also able to interact with aggrecan, with the exception of the mutant lacking the A domain, confirming that this ability resides entirely within this domain.

The large aggregating proteoglycan aggrecan is a major structural component of the extracellular matrix of articular cartilage. Recent cDNA cloning of the human aggrecan gene (AGC1) reveals a core protein of at least 2316 amino acids characterized by several distinct structural domains. Two globular domains, termed G1 and G2, are present at the amino terminus of the molecule and a third, termed G3, is present at the carboxy terminus. The G1 domain is homologous in structure to the cartilage link protein and accounts for the aggregating potential of aggrecan through its ability to interact with hyaluronic acid. The aggrecan gene is known to consist of 15 exons, with each exon encoding a distinct functional region of the mature protein. However, while the link protein gene is known to reside on chromosome 5 in the human, the location of the aggrecan gene is currently undetermined in any species. The probe (pAGG2) for the aggrecan gene was mapped on chromosome band 15q26, most likely in the subregion of 15q26.1, using fluorescence in situ hybridization. Clear signals were noted on both chromatids of chromosome band 15q26 in over 80% of the 300 metaphase cells examined in three independent experiments using pAGG2. No othermore » sites of hybridization were noted on both chromatids of any other chromosome band. The precise band location was identified by using chromsomes of about 650 bands and employing fluorescence reverse banding with chromomycin A3 and distamycin. 14 refs., 1 fig.« less

The chondrocytes in human articular cartilage from subjects of all ages express mRNAs for both of the aggregating proteoglycans aggrecan and versican, although the level of expression of versican mRNA is much lower than that of aggrecan mRNA. Aggrecan shows alternative splicing of the epidermal growth factor (EGF)-like domain within its C-terminal globular region, but there is no evidence for a major difference in situ in the relative expression of this domain with age. At all ages studied from birth to the mature adult, a greater proportion of transcripts lacked the EGF domain. The relative proportions of the two transcripts did not change upon culture and passage of isolated chondrocytes. In contrast, the neighbouring complement regulatory protein (CRP)-like domain was predominantly expressed irrespective of age, but cell culture did result in variation of the splicing of this domain. Versican possesses two EGF-like domains and one CRP-like domain, but at all ages the three domains were predominantly present in all transcripts. This situation persisted upon culture and passage of the chondrocytes. Thus, unlike aggrecan, the versican expressed by human articular cartilage does not appear to undergo alternative splicing of its C-terminal globular region, either in cartilage in situ or in chondrocytes in culture.

The mechanism of resistance to the alkylating agent melphalan was investigated in drug-sensitive and -resistant mutants of Chinese hamster ovary cells. Melphalan-resistant cells (MelR6), selected by a single exposure to melphalan, were 4.5-fold more resistant to drug than sensitive AUXB1 parental cells. Colchicine-resistant cells (CHRC5), which are cross-resistant to melphalan, were 15-fold more resistant than wild type cells. The kinetic parameters for drug influx were not significantly different in sensitive and resistant cells. The steady-state drug level in both MelR6 and CHRC5 cells was approximately 25 and 35% lower respectively than that in sensitive cells and this difference was accounted for by a more rapid rate of drug efflux from the resistant mutants. However, the level of drug resistance could not be explained entirely by this difference in drug transport. Sulfyddryl group levels were elevated in both MelR6 and CHRC5 cells relative to sensitive cells and these differences were statistically significant (P < 0.001). Furthermore, DNA interstrand cross-link formation was significantly lower in resistant cells than in sensitive cells. A similar rate of repair of DNA interstrand cross-links was observed in sensitive and resistant cells with the possible exception of a slower rate of repair in MelR6 cells. A higher level of DNA-protein cross-link activity, which may represent a mechanism for drug inactivation was observed in MelR6 cells. These studies suggest that resistance to melphalan in MelR6 and CHRC5 Chinese hamster ovary cell mutants is multifactorial involving lowered steady-state drug levels, enhanced drug efflux, elevated levels of sulfhydryl groups and decreased DNA interstrand cross-linking.