Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers.

Latent-transforming growth factor beta-binding protein 2 (LTBP-2) is a major component of arterial and lung tissue and of the ciliary zonule, the system of extracellular fibers that centers and suspends the lens in the eye. LTBP-2 has been implicated previously in the development of extracellular microfibrils, although its exact role remains unclear. Here, we analyzed the three-dimensional structure of the ciliary zonule in wild type mice and used a knockout model to test the contribution of LTBP-2 to zonule structure and mechanical properties. In wild types, zonular fibers had diameters of 0.5-1.0 micrometers, with an outer layer of fibrillin-1-rich microfibrils and a core of fibrillin-2-rich microfibrils. LTBP-2 was present in both layers. The absence of LTBP-2 did not affect the number of fibers, their diameters, nor their coaxial organization. However, by two months of age, LTBP-2-depleted fibers began to rupture, and by six months, a fully penetrant ectopia lentis phenotype was present, as confirmed by in vivo imaging. To determine whether the seemingly normal fibers of young mice were compromised mechanically, we compared zonule stress/strain relationships of wild type and LTBP-2-deficient mice and developed a quasi-linear viscoelastic engineering model to analyze the resulting data. In the absence of LTBP-2, the ultimate tensile strength of the zonule was reduced by about 50%, and the viscoelastic behavior of the fibers was altered significantly. We developed a harmonic oscillator model to calculate the forces generated during saccadic eye movement. Model simulations suggested that mutant fibers are prone to failure during rapid rotation of the eyeball. Together, these data indicate that LTBP-2 is necessary for the strength and longevity of zonular fibers, but not necessarily for their formation.

Protein interaction studies of MAGP-1 with tropoelastin and fibrillin-1.

Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.

Elastic fiber proteins in the glomerular mesangium in vivo and in cell culture.

BACKGROUND: Glomerular capillaries of the mammalian kidney are exposed to high intraluminal hydrostatic pressures and require elastic constraint to maintain size, shape, and integrity. Previous morphological and functional studies indicated that the extracellular matrices of glomeruli, that is, basement membrane and mesangial matrix, contribute to glomerular resilience and mechanical stability. Immunofluorescence microscopy findings demonstrated elastic fiber components to be located in the renal vasculature, including glomeruli. The aim of this study was to clarify the exact glomerular localization, composition, and cellular production of these proteins. METHODS: We examined the renal distribution of the elastic fiber proteins fibrillin-1, emilin, microfibril-associated glycoproteins (MAGPs) 1 and 2, latent transforming growth factor-binding protein-1 (LTBP-1), and elastin using immunohistology and immunoelectron microscopy of human, rat, and mouse kidneys. In mesangial cell cultures, we also studied the expression and extracellular deposition of such proteins by use of Northern blotting and immunocytochemistry. RESULTS: Fibrillin-1, emilin, MAGPs 1 and 2, and LTBP-1 were present in glomeruli of mouse, rat, and human kidney, where they were located predominantly in the mesangial extracellular matrix underlying glomerular endothelium and basement membrane. Several of these proteins, as well as elastin, were also expressed in the renal vasculature. While elastin localized to the glomerular vascular pole in afferent and efferent arterioles extending to Bowman's capsule, it was not found in the glomerular capillary tuft. Cultured mesangial cells of rat, mouse, and human kidneys expressed mRNAs of fibrillin-1, emilin, MAGP-2, and elastin, and the respective proteins localized within and outside of mesangial cells, as shown by immunocytochemistry. mRNA expression of fibrillin-1, emilin, and elastin was strong in quiescent mesangial cells; their gene expression was further up-regulated by transforming growth factor-beta1, while it was transiently reduced when cells were exposed to mitogenic 10% fetal calf serum and platelet-derived growth factor. CONCLUSIONS: These findings demonstrate that specific elastic fiber proteins are produced and secreted by mesangial cells. This process is regulated by growth factors. Their abundance in the extracellular matrix of the mesangium is in keeping with the concept that elastic fiber proteins contribute to the mechanical stability and elastic strength of the glomerular capillary tuft.

Effects of chronic prenatal ethanol exposure on hippocampal glutamate release in the postnatal guinea pig.

This study was designed to test the hypothesis that chronic prenatal ethanol exposure decreases basal and stimulated L-glutamate release in the hippocampus of young, postnatal guinea pigs. Timed, pregnant guinea pigs were randomly assigned to one of the following three chronic treatment groups: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose and pair-feeding to the ethanol group, and water. Each oral treatment was given daily throughout gestation. Spontaneous locomotor activity was increased on postnatal day (PD) 10, and brain and hippocampal weights were decreased on PD 12 in the offspring of the ethanol group compared with the isocaloric-sucrose/pair-fed and water groups. On PD 12, the 45 mM K(+)- and 10 microM veratridine-stimulated release of glutamate in transverse hippocampal slices was decreased in the ethanol group compared with the two control groups. This alteration in glutamate release produced by chronic prenatal ethanol exposure may decrease the efficiency of excitatory synaptic transmission in the hippocampus during postnatal life.

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells.

Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.

Developmental expression of dermatan sulfate proteoglycans in the elastic bovine nuchal ligament.

The nuchal ligament of bovines is a useful system in which to study elastic fibre formation since it contains up to 83% elastin and undergoes a period of rapid elastinogenesis during the last trimester of fetal development and in the first four post-natal months. To identify proteoglycans (PGs) which may be involved in this process we initially investigated changes in the glycosaminoglycan (GAG) profiles during nuchal ligament development. In contrast to the collagenous Achilles tendon, nuchal ligament exhibited: (a) elevated hyaluronan (HA) levels in the peak period of elastin-associated microfibril (fibrillin) synthesis (130-200 days) which precedes elastinogenesis; and (b) markedly increased synthesis of a glucuronate-rich copolymeric form of dermatan sulfate (DS) in the period corresponding to elastin formation (200-270 days). Analysis of DSPGs isolated from 230-day nuchal ligament showed that this copolymer was predominantly associated with a glycoform of biglycan which was specifically elevated at this stage in development. This finding was consistent with Northern blot analysis which showed that steady-state biglycan mRNA levels increased significantly during the elastinogenic period. In contrast, the mRNA levels for decorin, the only other DSPG detected in this tissue, declined rapidly after 140 days of fetal development. In conclusion, the results suggest that HA may play a role in microfibril assembly and that a specific glycoform of biglycan may be associated with the elastinogenic phase of elastic fibre formation.

Effects of chronic prenatal ethanol exposure on locomotor activity, and hippocampal weight, neurons, and nitric oxide synthase activity of the young postnatal guinea pig.

Decreased nitric oxide synthase (NOS)-catalyzed formation of NO from L-arginine may be involved in ethanol teratogenesis involving the hippocampus. This hypothesis was tested by determining the effects of chronic prenatal ethanol exposure on locomotor activity and on hippocampal weight, number of CA1 and CA3 pyramidal cells and dentate gyrus granule cells, and NOS activity of the postnatal guinea pig. Timed, pregnant guinea pigs received one of the following chronic oral regimens throughout gestation: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose/pair-feeding, or water. At postnatal day (PD) 10, spontaneous locomotor activity was measured. At PD 12, histological analysis was performed on the hippocampal formation, in which hippocampal CA1 and CA3 pyramidal cells and dentate gyrus granule cells were counted; body, brain, and hippocampal weights were measured; and hippocampal NOS enzymatic activity was determined using a radiometric assay. Chronic prenatal ethanol exposure produced hyperactivity, decreased the brain and hippocampal weights with no change in body weight, decreased the number of hippocampal CA1 pyramidal cells by 25-30%, and had no effect on hippocampal NOS activity compared with the two control groups. These data, together with our previous findings in the fetal guinea pig, demonstrate that chronic prenatal ethanol exposure decreases hippocampal NOS activity in near-term fetal life that temporally precedes the selective loss of hippocampal CA1 pyramidal cells in postnatal life.

Strain-induced symmetry breakdown in HOLZ lines from L1(2) titanium tri-aluminides.

Energy filtered CBED patterns of the Cr-stabilized L1(2) phase of a titanium tri-aluminide alloy reveal deficit higher order Laue zone (HOLZ) lines in the zeroth order diffraction disk, for which the expected 4-mm symmetry is significantly broken in the <001> projection. This apparent break of symmetry may be explained by the presence of lattice strains of order 10(-3). Effects of strain-induced lattice distortions on HOLZ line symmetries are calculated by an introduction of rhombohedral, tetragonal or monoclinic distortions to the cubic unit cell. It is shown how tensile and shear components of strain affect the overall HOLZ line symmetries in different ways.

Molecular cloning of a novel human PAPS synthetase which is differentially expressed in metastatic and non-metastatic colon carcinoma cells.

Subtractive hybridisation was used to select for genes which are differentially expressed between a highly metastatic human colon carcinoma cell line, KM12SM, and the isogenetic non-metastatic cell line, KM12C. This led to the isolation of cDNA clones for a novel human adenosine 5'-phosphosulphate kinase/ATP sulphurylase (PAPS synthetase). Northern hybridisation revealed a single 4.2 kb mRNA species which showed an approximately 20-fold higher level of expression in the non-metastatic cell line than in the metastatic cell line. The overlapping cDNA clones together covered 3,774 bp including the entire coding region of 1,842 bp encoding a protein of 614 amino acids (calculated molecular mass of 69,496 Da). The protein contains consensus sequences for APS kinase and ATP sulphurylase, in its amino- and carboxy-terminal regions, respectively, as well as other sequences that are highly conserved amongst ATP sulphurylases and APS kinases. Interestingly, consensus sequences for GTPase activity were also identified, indicating that enzyme activity may be regulated by an intrinsic GTPase mechanism. Overall the new protein is 78% homologous with a previously described human PAPS synthetase (PAPSS1) indicating that we have identified the second member of a gene family which we have provisionally named PAPSS2. The gene locus for PAPSS2 was identified on chromosome 10 at 10q23.1-q23.2. This locus has synteny with the mouse brachymorphic gene recently identified as a PAPS synthetase (SK2). PAPSS2 appears to be the human homologue of this gene and thus PAPSS2 is likely to be important in human skeletogenesis.

Microfibril-associated glycoprotein-2 specifically interacts with a range of bovine and human cell types via alphaVbeta3 integrin.

Microfibril-associated glycoprotein (MAGP)-1 and MAGP-2 are small structurally related glycoproteins that are specifically associated with fibrillin-containing microfibrils. MAGP-2, unlike MAGP-1, contains an RGD motif with potential for integrin binding. To determine if the RGD sequence is active, a series of cell binding assays was performed. MAGP-2 was shown to promote the attachment and spreading of bovine nuchal ligament fibroblasts when coated onto plastic wells in molar quantities similar to those of fibronectin. In contrast, approximately 10-fold more MAGP-1 was required to support comparable levels of cell adhesion. The fibroblast binding to MAGP-2 was completely inhibited if the peptide GRGDSP or the MAGP-2-specific peptide GVSGQRGDDVTTVTSET was added to the reaction medium at a 10 microM final concentration. The control peptide GRGESP had no effect on the interaction. These findings indicate that the cell interaction with MAGP-2 is an RGD-mediated event. A monoclonal antibody to human alphaVbeta3 integrin (LM609) almost completely blocked cell attachment to MAGP-2 when added to the medium at 0.5 microgram/ml, whereas two monoclonal antibodies specific for the human beta1 integrin subunit, 4B4 (blocking) and QE2.E5 (activating), had no effect even at 10 microgram/ml. Fetal bovine aortic smooth muscle cells, ear cartilage chondrocytes, and arterial endothelial cells and human skin fibroblasts and osteoblasts were also observed to adhere strongly to MAGP-2. In addition, each cell type was able to spread on MAGP-2 substrate, with the exception of the endothelial cells, which remained spherical after 2 h of incubation. The binding of each cell type was blocked when the anti-alphaVbeta3 integrin antibody was included in the assay, indicating that alphaVbeta3 integrin is the major receptor for MAGP-2 on several cell types. Thus, MAGP-2 may mediate interactions between fibrillin-containing microfibrils and cell surfaces during the development of a variety of tissues.

47,49Ti NMR: hyperfine interactions in oxides and metals.

A 47,49Ti NMR characterisation is given of various polymorphs of TiO2 (anatase, rutile and brookite), Ti2O3, perovskites CaTiO3 and BaTiO3, FeTiO3, TiB2, titanium metal, the titanium aluminides Ti3Al, TiAl, TiAl2, TiAl3, and TiAg. Values of chemical or Knight shift, nuclear quadrupole coupling constant and asymmetry parameter were derived from the (1/2, -1/2) powder lineshapes. For TiB2, titanium metal, TiAl, and TiAl3, where +/- (1/2, 3/2), and higher satellite transitions were observed, a value for the axial component of the Knight shift was obtained.

The exon structure of the human MAGP-2 gene. Similarity with the MAGP-1 gene is confined to two exons encoding a cysteine-rich region.

A cDNA for human microfibril-associated glycoprotein-2 (MAGP-2) was used to screen a human leukocyte genomic DNA library in EMBL-3 vector. One clone, clone H (10 kilobase pairs (kbp)), was isolated that contained most of the MAGP-2 gene. The remainder of the 3' end of the gene was obtained by direct polymerase chain reaction amplification of genomic DNA. The human MAGP-2 gene was found to be about 11 kbp in size and to contain 10 evenly distributed exons. The internal exons range in size from 30 base pairs (bp) to 88 bp with exons 4 and 6 the only exons of equal size (45 bp). All internal intron:exon junctions are defined by canonical splice donor and acceptor sites. Each junction has a 1/2 codon split with the exception of the exon 8/9 junction, which has a 2/1 split. The translation initiation codon is in exon 2, and the final exon contains 110 bp of coding sequence, including 2 cysteine codons. Primer extension experiments identified only one major transcription initiation site, 213 bases upstream of the ATG site. Rapid analysis of cDNA ends-polymerase chain reaction analysis of the 5' end of MAGP-2 mRNA from placenta confirmed this result and did not detect any alternative splicing of transcripts. The putative promoter region of the MAGP-2 gene was found to be AT-rich and it lacked a TATA box and other common regulatory elements. However the sequence surrounding the transcription start site CTCA(+1)TTCC was similar to the consensus CTCA(+1)NTCT (N is any nucleoside) for an initiator element found in terminal deoxynucleotidyltransferase and a number of other highly regulated genes. Comparison with the previously characterized human MAGP-1 gene showed that structural similarity was largely confined to the exact size, sequence, and junction alignment of the two penultimate exons which encode the first six of the seven cysteine residues that are precisely spaced in both proteins. The findings are consistent with the growing evidence that, although MAGP-1 and MAGP-2 are both intimately involved in the biology of fibrillin-containing microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif.

Preferential pre-mRNA utilisation of an upstream cryptic 5' splice site created by a single base deletion mutation in exon 37 of the FBN-1 gene.

A heterozygous deletion of a single base (A4704) from exon 37 of the fibrillin-1 gene was defined in a patient with Marfan syndrome and subsequently in his previously undiagnosed father. The deletion created a cryptic 5' splice site in exon 37 which was utilised in preference to the normal 5' splice site during pre-mRNA processing in skin fibroblasts cultured from the proband. The mutant mRNA showed a 48-bp deletion from the 3' end of exon 37 which was predicted to restore the reading frame in the mutant mRNA and result in the deletion of a 16-amino-acid sequence from a central eight-cysteine repeat motif of the fibrillin-1 molecule. Interestingly, the cryptic 5' splice site in exon 37 and the normal 5' splice site had equally strong consensuses for splice-site selection. The preferential utilisation of the cryptic site is discussed in relation to current theories on the mechanisms involved in pre-mRNA splicing. Analysis by reverse-transcription PCR indicated that, in the patients skin fibroblasts, the steady-state level of the mis-spliced mutant mRNA was close to that from the normal allele. In addition, evidence from immunoblotting and pulse-chase biosynthetic labelling indicated that close to normal amounts of fibrillin-1 were being synthesised and secreted by the cells. However, in contrast to control cells cultured from an unaffected individual, little fibrillin-1 was detected, either biosynthetically or by immunofluorescence, in the extracellular matrix produced by the proband's fibroblasts. Thus, the slightly shorter mutant fibrillin-1 molecules appeared to be exerting a powerful dominant-negative effect on the incorporation of normal fibrillin-1 molecules into microfibrils in this culture system. This severe inhibition of microfibril synthesis in cell culture contrasts with the 'classic' phenotype of the proband, suggesting that factors influencing microfibril formation may differ greatly between in vivo and in vitro environments.

Microfibril-associated glycoprotein-2 (MAGP-2) is specifically associated with fibrillin-containing microfibrils but exhibits more restricted patterns of tissue localization and developmental expression than its structural relative MAGP-1.

We developed an affinity-purified anti-MAGP-2 peptide antibody that specifically identified MAGP-2 on Western blots of purified matrix proteins and extracts of nuchal ligament. Immunolocalization studies on tissues from a 210-day-old fetus and a mature bovine showed that MAGP-2 was located in similar regions to MAGP-1 and fibrillin-1 but that the distribution of MAGP-2 was more restricted. In fetal nuchal ligament, skeletal muscle, and spleen the distribution of MAGP-2 was indistinguishable from that of MAGP-1. In contrast to MAGP-1, MAGP-2 was not detected in the medial layer of fetal thoracic aorta and in much of the peritubular matrix of fetal and mature kidney and in the mature ocular zonule. Some differences in the immunolocalization patterns were also evident in fetal lung, cartilage, skin, and heart. Immunoelectron microscopy confirmed that MAGP-2 was specifically associated with fibrillin-containing microfibrils in nuchal ligament, dermis, adventitia of aorta, glomerular mesangium and perimysium. Northern blotting of RNA from tissues of a 210-day-old fetus indicated that steady-state MAGP-2 mRNA levels were highest in nuchal ligament. Significant expression was also detected in lung, heart, skeletal muscle, skin, and Achilles tendon. The tissue pattern of MAGP-2 expression differed significantly from that of MAGP-1. MAGP-2 expression appeared to be higher in nuchal ligament, heart, and skeletal muscle and lower in aorta and kidney. In nuchal ligament, MAGP-2 mRNA expression appeared to peak around 180 days of fetal development, which correlates with the period of onset of elastinogenesis in this tissue. Overall, the immunolocalization and expression patterns of MAGP-2 appeared to be distinct from those of other microfibrillar components. This is consistent with the view that MAGP-2 plays a unique role in the biology of the microfibrils, perhaps by mediating their interaction with cell surfaces at specific stages of development and differentiation. (J Histochem Cytochem 46:871-885, 1998)

Direct right ventricular puncture for hemodynamic evaluation of a mechanical tricuspid valve prosthesis: a new indication for an old procedure.

The advent of transvenous right heart catheterization has relegated direct transthoracic right ventricular puncture largely to the role of "interesting historical footnote." However, in the case of a right ventricle that is "protected" by a mechanical tricuspid valve prosthesis, direct right ventricular puncture represents a reasonable alternative for obtaining accurate hemodynamic information.

Microfibril-associated glycoprotein-1 (MAGP-1) binds to the pepsin-resistant domain of the alpha3(VI) chain of type VI collagen.

The interactions of type VI collagen have been investigated, using solid phase binding assays, with two components of the fibrillin-containing microfibrils, the elastin-binding protein, MAGP-1 and its structural relative MAGP-2. Both native and pepsin-treated forms of type VI collagen specifically bound to MAGP-1 but not to MAGP-2. Pepsin type VI collagen was shown to block the binding of MAGP-1 to native type VI collagen indicating that the major MAGP-1-binding site was in the triple-helical region of the molecule. MAGP-1 was found not to bind to collagens I, III, and V. Affinity blotting of pepsin-treated type VI collagen showed that MAGP-1 binding was specific for the collagenous domain of the alpha3(VI) chain. Decorin and biglycan were found not to inhibit the interaction of pepsin-treated type VI collagen with MAGP-1, indicating that its binding site on the collagen is not close to that for the proteoglycans. Reduction and alkylation of disulfide bonds in MAGP-1 did not destroy its type VI collagen-binding properties, indicating that the binding site was likely to be in the cysteine-free, N-terminal domain of MAGP-1. Interestingly, the interaction of MAGP-1 with type VI collagen was inhibited by tropoelastin, suggesting that the binding sites for tropoelastin and type VI collagen may be in the same domain of MAGP-1. A peptide, corresponding to amino acids 29-38 of MAGP-1, was found to inhibit the interactions of MAGP-1 with type VI collagen and tropoelastin. The results suggest that the peptide may contain the binding sequences for both type VI collagen and tropoelastin, and thus that these two proteins may share the same binding site on MAGP-1. The interactions of MAGP-1 with type VI collagen and tropoelastin were both determined to be of moderately high affinity, with Kd values of 5.6 x 10(-7) M and 2.6 x 10(-7) M, respectively. The findings indicate that MAGP-1 may mediate a molecular interaction between type VI collagen microfibrils and fibrillin-containing microfibrils, structures which are often found in close proximity to each other in a wide range of extracellular matrices.

Immunohistochemical and ultrastructural localization of MP78/70 (betaig-h3) in extracellular matrix of developing and mature bovine tissues.

MP78/70 is a matrix protein, with 78-kD and 70-kD isoforms, which was initially identified in bovine tissue extracts designed to solubilize elastin-associated microfibrils. Peptide analysis has shown that MP78/70 is closely related to the human protein, betaig-h3. In the present study an antibody raised to a synthetic betaig-h3 peptide was shown specifically to identify MP78/70 in purified form and in bovine tissue extracts. This is consistent with MP78/70 and betaig-h3 being the bovine and human forms, respectively, of the same protein. The antibody was further affinity-purified on MP78/70 bound to Sepharose and used to localize the protein in a range of bovine tissues. Immunofluorescence showed that MP78/70 was localized to collagen fibers in tissues such as developing nuchal ligament, aorta and lung, and mature cornea; to reticular fibers in fetal spleen; and to capsule and tubule basement membranes in developing kidney. No general localization to elastic fibers was observed. The staining pattern in most tissues more closely resembled that of Type VI collagen, which occurs as collagen fiber-associated microfibrils, than that of fibrillin-1, a component of elastin-associated microfibrils. However, MP78/70 appeared to be less widely distributed than Type VI collagen. Immunoelectron microscopy showed that MP78/70 was predominantly found in loose association with collagen fibers in most tissues examined and was also located on the surface of the capsule basement membrane in developing kidney. Double labeling experiments indicated that MP78/70 is co-distributed with Type VI collagen microfibrils located in these regions. In some elastic tissues significant immunolabel was detected in regions of interface between collagen fibers and fibrillin-containing microfibrils of adjacent elastic fibers, and at the outer margins of the latter structures. Overall, the evidence points to MP78/70 having a bridging function, perhaps in association with Type VI collagen microfibrils, linking or stabilizing the interaction between interstitial collagen fibrils and other matrix structures, including some basement membranes and elastin-associated microfibrils.

Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure for fibrillin-containing microfibrils as visualized by the rotary shadowing technique.

This study used immunoelectron microscopic techniques to define the ultrastructural location of MAGP-1 on the fibrillin-containing microfibrils of the ocular zonule. A specific anti-MAGP-1 monoclonal antibody (MAb), 11B, was produced that did not crossreact with fibrillin-1 or other microfibrillar proteins. MAb 11B was shown by immunofluorescence to localize intensely to zonular tissue. Postembedding immunoelectron microscopy showed that MAGP-1 was associated with microfibrils throughout the zonule, with the exception of a narrow band of microfibrils at the junction with the lens capsule. With preembedding labeling, the anti-MAGP-1 MAb was found to localize in a crossbanding pattern, at intervals of about 50 nm, to microfibrils throughout the zonule and along bundles of microfibrils in surrounding vitreous tissue. Rotary shadowing of isolated microfibrils showed a "beads on a string" morphology with a periodicity of about 50 nm. With immunogold labeling, the anti-MAGP-1 antibody specifically localized on the beads in a symmetrical manner. Occasionally two gold partides were attached to the same bead, suggesting that multiple MAGP-1 molecules were present in the structure. The results indicate that MAGP-1 is intimately and regularly associated with the bead regions of fibrillin-containing microfibrils. The findings are consistent with a major structural role for MAGP-1 in microfibril biology.

Co-ordinating care for Canadian travellers.

Case management is a popular term these days. It refers to caring for the whole patient and his or her family by incorporating all necessary resources available within the community.

Further characterization of proteins associated with elastic fiber microfibrils including the molecular cloning of MAGP-2 (MP25)

Together with the 31-kDa microfibril-associated glycoprotein (MAGP), four polypeptides designated MP340 (340 kDa), MP78 (78 kDa), MP70 (70 kDa), and MP25 (25 kDa) have previously been identified in tissue extracts designed specifically to solubilize the microfibrillar component of elastic fibers. In the present study, both MP78 and MP70 were shown to be forms of a protein which is closely related to the human protein beta ig-h3, and MP340 was confirmed to be the bovine form of fibrillin-1. Peptide sequences from MP25 proved to be unique, and affinity-purified anti-MP25 antibodies were shown, by immunofluorescence and immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. This confirmed that MP25 was a distinct component of these structures. Expression screening of nuchal ligament cDNA libraries yielded a cDNA, cM10A (770 base pairs) which encodes amino acid sequences matching those of the MP25 peptides. Further library screening with cM10A identified cDNAs which encode the complete primary structures of bovine and human MP25. Bovine and human MP25 were found to be around 80% homologous and contain 170 and 173 amino acids, respectively. Data base searches revealed that MP25 had significant similarity of structure only with MAGP, indicating that the two proteins form a new family of microfibrillar proteins. In acknowledgment, MP25 has been formally renamed MAGP-2, and MAGP is referred to as MAGP-1. The close similarity between the two proteins (57%) is confined to a central region of 60 amino acids where there is precise alignment of 7 cysteine residues. Elsewhere the MAGP-2 molecule is rich in serine and threonine residues and contains an RGD motif. MAGP-2 lacks the proline-, glutamine-, and tyrosine-rich sequences and a hydrophobic carboxyl terminus, characteristic of MAGP-1. These structural differences suggest that MAGP-2 has some functions which are distinct from those of MAGP-1. The locus of the human MAGP-2 gene was identified on chromosome 12 in the region of 12p12.3-12p13.1.