Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-␣1(V) chains were created by homologous recombination. The col5a1؊/؊ animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1؉/؊ animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.Type V collagen is a member of the fibril subclass of collagens, which have in common a triple helical domain composed of an uninterrupted series of Gly-X-Y triplets. Type V collagen is a quantitatively minor component of predominantly type I collagen fibrils in most non-cartilaginous tissues. Several isoforms of type V collagen exist, which differ in the type and ratio of constituent chains, including heterotypic molecules containing type XI collagen chains. The most abundant and widely distributed isoform is ␣1(V) 2 ␣2(V), which forms heterotypic fibrils with type I collagen (1). The role of type V collagen in the organization and biological properties of collagenous extracellular matrix is poorly understood. Observations of an inverse correlation between type V collagen:type I collagen ratios and collagen fibril diameter in in vitro fibril assembly experiments (2), cell cultures (3, 4), and in various tissues (5) have led to the hypothesis that type V collagen serves as a negative regulator of collagen fibril diameter (3-5). That function may be mediated by retention of the non-collagenous amino-terminal propeptide after type V collagen molecules are incorporated into fibrils (2, 6 -9). This non-collagenous domain projects outward through the gap between adj...

). Strains carrying one wild-type and two mutant alleles of the three loci were constructed to study the kinetics and specificity of ion transport of each system in isolation. The transport systems had different Km Assay of Mg2+ transport. Uptake using 28Mg2' has been described previously (3). Mg2+ uptake was measured in strains of S. typhimurium that carry one wild-type and two mutant alleles of the three genes corA, mgtA, and mgtB (2). This allowed dissection of the properties of each individual transport system without interference from the others. For clarity, the Mg2+ transport system present is referred to as CorA, MgtA, or MgtB rather than by the longer genotypic designation of defective systems, e.g., as the CorA system rather than as corA+ mgtA mgtB. The phenotype of each of the strains carrying only a single Mg2+ transport system was always verified the day before each transport assay by using a disk sensitivity test to determine the pattern of growth inhibition by divalent cations, a pattern that is characteristic for each strain (2). The apparent Ki for inhibition by other divalent cations was determined from the cation concentration giving 50% inhibition (IC50) (Fig. 1)

Salmonella typhimurium strains lacking the CorA Mg2+ transport system retain Mg2+ transport and the ability to grow in medium containing a low concentration of Mg2+. Mutagenesis of a corA strain followed by ampicillin selection allowed isolation of a strain that required Mg2+-supplemented media for growth. This strain contained mutations in at least two loci in addition to corA, designated mgtA and mgtB (for magnesium transport). Strains with mutations at all three loci (corA, mgtA, and mgtB) exhibited no detectable Mg2+ uptake and required 10 mM Mg2+ in the medium for growth at the wild-type rate. A wild-type allele at any one of the three loci was sufficient to restore both Mg2+ transport and growth on 50 ,uM Mg2+. P22 transduction was used to map the mgt loci. The mgtA mutation was located to approximately 98 map units (cotransducible with pyrB), and mgtB mapped at about 80.5 map units (near gltC). A chromosomal library from S. typhimurium was screened for clones that complemented the Mg2+ requirement of a corA mgtA mgtB mutant. The three classes of plasmids obtained could each independently restore Mg2+ transport to this strain and corresponded to the corA, mgtA, and mgtB loci. Whereas the corA locus of S. typhimurium is analogous to the corA locus previously described for Escherichia coli, neither of the mgt loci described in this report appears analogous to the single mgt locus described in E. coli. Our data in this and the accompanying papers (M. D. Snavely, J. B. Florer, C. G. Miller, and M. E. Maguire, J. Bacteriol. 171:4752-4760, 4761-4766, 1989) indicate that the corA, mgtA, and mgtB loci of S. typhimurium represent three distinct systems that transport Mg2>

Mesenchymal stem cells (MSCs) have been used to repair connective tissue defects in several animal models. Compared to "natural healing" controls (no added cells), MSC-collagen gel constructs in rabbit tendon defects significantly improve repair biomechanics. However, ectopic bone forms in 28% of MSC-treated rabbit tendons. To understand the source of bone formation, three studies were performed. In the first study, the hypothesis was tested that MSCs delivered during surgery contribute to bone formation in the in vivo repair site. Adjacent histological sections in the MSC-treated repair tissue were examined for pre-labeled MSCs and for cells showing positive alkaline phosphatase (ALP) activity. Both cells were observed in serial sections in regions of ectopic bone. Contralateral "natural healing" tendons lacked both markers. In the other two studies, the effects of osteogenic supplements and construct geometry (monolayer vs. 3-D) on ALP activity were studied to test three hypotheses: that rabbit MSCs increase ALP activity over time in monolayer culture conditions; that adding osteogenic inducing supplements to the culture medium increases cellular protein in monolayer culture; and that rabbit MSCs increase ALP activity both in monolayer and in 3-D constructs, with and without media supplements. Culture in monolayer under similar conditions to in vivo (as in the first study) did not increase ALP at 2 or 4 weeks. Medium designed to increase osteogenic activity significantly increased cell numbers (cellular protein increased by 260%) but did not affect ALP activity either in monolayer or 3 -0 constructs (p > 0.12). However, MSCs in 3-D constructs exhibited higher ALP activity than cells in monolayer, both in the presence (p < 0.005) and absence of supplement (p < 0.045). These results suggest that in vitro conditions may critically influence cell differentiation and protein expression.Mechanisms responsible for these effects are currently under investigation.

Collagens V and XI comprise a single regulatory type of fibrilforming collagen with multiple isoforms. Both co-assemble with collagen I or II to form heterotypic fibrils and have been implicated in regulation of fibril assembly. The objective of this study was to determine the roles of collagens V and XI in the regulation of tendon fibrillogenesis. Flexor digitorum longus tendons from a haplo-insufficient collagen V mouse model of classic Ehlers Danlos syndrome (EDS) had decreased biomechanical stiffness compared with controls consistent with joint laxity in EDS patients. However, fibril structure was relatively normal, an unexpected finding given the altered fibrils observed in dermis and cornea from this model. This suggested roles for other related molecules, i.e. collagen XI, and compound Col5a1 ؉/؊ ,Col11a1 ؉/؊ tendons had altered fibril structures, supporting a role for collagen XI. To further evaluate this, transcript expression was analyzed in wild type tendons. During development (E18-P10) both collagen V and XI were comparably expressed; however, collagen V predominated in mature (P30) tendons. The collagens had a similar expression pattern. Tendons with altered collagen V and/or XI expression (Col5a1؊/؊ ) were analyzed at E18. All genotypes demonstrated a reduced fibril number and altered structure. This phenotype was more severe with a reduction in collagen XI. However, the absence of collagen XI with a reduction in collagen V was associated with the most severe fibril phenotype. The data demonstrate coordinate roles for collagens V and XI in the regulation of fibril nucleation and assembly during tendon development.Abnormal collagen fibril formation is characteristic of the classic form of Ehlers-Danlos syndrome (EDS).2 Patients with classic EDS (types I and II) have a broad spectrum of generalized connective tissue defects including hyper-extensible skin, fragile skin with wide, depressed, callused scarring, inguinal hernias, and rectal prolapse as well as aortic root dilation and valve prolapse (1, 2). In addition, laxity in the joints, leading to instability and easy dislocation as well as joint hyper-extensibility, is a characteristic feature resulting from dysfunctional tendons and ligaments. More than half of all instances of classic EDS have been linked to heterozygous mutations in the genes for collagen V (3-13). The most common mutation type in classic EDS is one that results in a functional loss of one Col5a1 allele (14, 15).Collagen V is a fibril-forming collagen. The fibril-forming collagen subfamily includes collagens I, II, III, V, XI, XXIV, and XXVII, and the genes cluster into three distinct clades (16). Collagens I, II, and III are the major components of all collagen fibrils. Collagens V and XI are quantitatively minor collagens found as heterotypic fibrils with collagens I, II, and III and have a regulatory function in fibrillogenesis (17). Collagens XXIV and XXVII have structural differences relative to collagens I, II III, V, and XI, and their specific roles remain to be elucida...