ABSTRACT
This work was designed to evaluate the efficacy of a postbiotic compound produced by stabilized non-viable Lactobacilli on the health, growth performance, immunity, and gut status against Escherichia coli (E. coli) challenge of broiler chickens. A total of 400, day-old broiler chicks were allocated into 4 equal groups (1-4) consisting of 100; each assigned into 2 equal replicates (50 each). Chickens in the 1st group were received the dry form of the compound at doses of 1 kg and 0.5 kg/ton feed for starter and grower, and the finisher diets, respectively. Chickens in the 2nd group were given the aqueous form of the compound in a dose of 4 mL/L of the drinking water during the first 3 days of life and at a day before and after each vaccination. Feed and water treatment regimens were administered to chickens in the 3rd group. Group 4 was kept without treatment. Each bird in the 1st, 2nd, 3rd, and 4th group was challenged with E. coli (O78) at 1-week-old. All groups were kept under observation till 5-week-old. Statistical analysis included one-way ANOVA and other methods as described with significant differences at P ≤ 0.05. The results indicated that feed and water treatments with the postbiotic compound induced more significant (P ≤ 0.05) amelioration of a disease picture, enhancement of growth performance, boosting of immune response, improvement of bursa of Fabricius/body weight ratio, and reduction of intestinal coliform count in challenged chickens when compared with challenged non-treated chickens. In conclusion, the postbiotic compound either in a dry and/or an aqueous form is recommended for improving the health, performance, and immunity of colisepticaemic broiler chickens.
Subject(s)
Chickens , Escherichia coli Infections , Animal Feed/analysis , Animals , Diet/veterinary , Dietary Supplements , Escherichia coli/physiology , Escherichia coli Infections/prevention & control , Escherichia coli Infections/veterinary , LactobacillusABSTRACT
The aim of this paper is to illustrate a rare case of primary aortoenteric fistula in the presence of disseminated infection and review the critical decision-making process. A clinical case was reviewed for its initial presentation, planning of treatment strategy and outcome. A secondary literature search for discussion on current accepted recommendations for primary aortoenteric fistula was then completed. Aortoenteric fistulas are rare pathologies with highly morbid potential. Their diagnosis requires a high index of suspicion and prompt intervention is critical to patient survival. In conclusion, aortoenteric fistula most commonly arises from large atherosclerotic aneurysms but can be caused by systemic infection. In the case of the latter, extra-anatomic repair appears to be the treatment of choice.
Subject(s)
Aneurysm, Infected/microbiology , Antineoplastic Agents/adverse effects , Aortic Aneurysm, Abdominal/microbiology , Aortic Diseases/microbiology , BCG Vaccine/adverse effects , Duodenal Diseases/microbiology , Fistula/microbiology , Intestinal Fistula/microbiology , Mycobacterium bovis/pathogenicity , Tuberculosis/microbiology , Administration, Intravesical , Aneurysm, Infected/diagnosis , Aneurysm, Infected/surgery , Anti-Bacterial Agents/therapeutic use , Antineoplastic Agents/administration & dosage , Antitubercular Agents/therapeutic use , Aortic Aneurysm, Abdominal/diagnosis , Aortic Aneurysm, Abdominal/surgery , Aortic Diseases/diagnosis , Aortic Diseases/surgery , Aortography , BCG Vaccine/administration & dosage , Blood Vessel Prosthesis Implantation , Cephalosporins , Debridement , Duodenal Diseases/diagnosis , Duodenal Diseases/surgery , Duodenoscopy , Fistula/diagnosis , Fistula/surgery , Gastroscopy , Humans , Intestinal Fistula/diagnosis , Intestinal Fistula/surgery , Male , Middle Aged , Tomography, X-Ray Computed , Treatment Outcome , Tuberculosis/diagnosis , Tuberculosis/drug therapy , Urinary Bladder Neoplasms/drug therapyABSTRACT
The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.
Subject(s)
Microfilament Proteins/genetics , Mutation/genetics , Scleroderma, Systemic/congenital , Scleroderma, Systemic/genetics , Skin/pathology , Biopsy , Cell Adhesion , Cell Movement , Collagen/metabolism , DNA Mutational Analysis , Elastin/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Family , Female , Fibrillin-1 , Fibrillins , Humans , Immunohistochemistry , Male , Mesoderm/pathology , Microfibrils/metabolism , Microfibrils/pathology , Microfilament Proteins/metabolism , Pedigree , Phenotype , Scleroderma, Systemic/pathology , Signal Transduction , Skin/ultrastructure , Syndrome , Transforming Growth Factor beta/metabolismABSTRACT
Fibrillins are the structural components of extracellular microfibrils that impart physical properties to tissues, alone or together with elastin as elastic fibers. Genetic studies in mice have revealed that fibrillin-rich microfibrils are also involved in regulating developmental programs and homeostatic processes through the modulation of TGF-beta/BMP signaling events. A new paradigm has thus emerged whereby the spatiotemporal organization of microfibrils dictates both the cellular activities and physical properties of connective tissues. These observations have paved the way to novel therapeutic approaches aimed at counteracting the life-threatening complications in human conditions caused by dysfunctions of fibrillin-rich microfibrils.
Subject(s)
Cardiovascular Diseases/metabolism , Extracellular Matrix/metabolism , Microfibrils/metabolism , Morphogenesis , Animals , Extracellular Matrix/chemistry , Fibrillins , Humans , Microfibrils/chemistry , Microfilament Proteins/chemistry , Microfilament Proteins/metabolismSubject(s)
Cell Shape/physiology , Connective Tissue/physiology , Microfibrils/physiology , Microfilament Proteins/physiology , Signal Transduction/physiology , Animals , Fibrillins , Humans , Marfan Syndrome/etiology , Marfan Syndrome/genetics , Marfan Syndrome/physiopathology , Mice , Mice, Mutant Strains , Microfilament Proteins/genetics , Mutation/geneticsABSTRACT
BACKGROUND: Fibulin-5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin-5 in human skin and its changes during the ageing process are not known. OBJECTIVES: To explore the involvement of fibulin-5 in skin ageing, the age-dependent changes in fibulin-5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin-1 and fibulin-2. Methods The distribution of elastin, fibrillin-1, fibrillin-2, fibulin-2 and fibulin-5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B-irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. RESULTS: In the reticular dermis of young sun-protected skin from the upper arm, fibulin-5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin-5 showed candelabra-like structures perpendicular to the epidermis with an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin-1. Fibulin-5 in the reticular dermis decreased and disappeared with age even in sun-protected skin from the thigh, abdomen and upper arm. In sun-exposed skin, fibulin-5 was extremely reduced in the dermis of cheek skin even from a 20-year-old man. UVB irradiation reduced fibulin-5, fibulin-2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin-5 was increased in solar elastosis, like that of other elastic fibre components. CONCLUSIONS: These results suggest that fibulin-5 is a good marker of skin ageing and that the earlier loss of fibulin-5 may involve age-dependent changes in other elastic fibre components.
Subject(s)
Connective Tissue Diseases/metabolism , Dermis/chemistry , Extracellular Matrix Proteins/analysis , Recombinant Proteins/analysis , Skin Aging/physiology , Ultraviolet Rays/adverse effects , Adolescent , Adult , Aged , Biomarkers/analysis , Calcium-Binding Proteins/analysis , Child , Child, Preschool , Dermis/radiation effects , Elastin/analysis , Female , Fibrillin-1 , Fibrillin-2 , Fibrillins , Humans , Immunohistochemistry/methods , Male , Microfilament Proteins/analysis , Middle AgedABSTRACT
Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.
Subject(s)
Cartilage/metabolism , Extracellular Matrix Proteins/metabolism , Glycoproteins/metabolism , Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Osteoarthritis/metabolism , Osteochondrodysplasias/metabolism , Aggrecans , Calcium-Binding Proteins/metabolism , Calnexin , Carrier Proteins/metabolism , Cartilage/pathology , Cartilage/ultrastructure , Cartilage Oligomeric Matrix Protein , Child , Chondroitin Sulfate Proteoglycans/metabolism , Collagen/metabolism , DNA Mutational Analysis , Decorin , Endoplasmic Reticulum, Rough/metabolism , Endoplasmic Reticulum, Rough/ultrastructure , Extracellular Matrix Proteins/genetics , Female , Fibromodulin , Glycoproteins/genetics , HSP47 Heat-Shock Proteins , Humans , Inclusion Bodies/pathology , Inclusion Bodies/ultrastructure , Intracellular Fluid/metabolism , Lectins, C-Type , Matrilin Proteins , Osteoarthritis/pathology , Osteochondrodysplasias/pathology , Protein Disulfide-Isomerases/metabolism , Proteoglycans/metabolismABSTRACT
To elucidate the contribution of the extracellular microfibril-elastic fiber network to vertebrate organogenesis, we generated fibrillin 2 (Fbn2)-null mice by gene targeting and identified a limb-patterning defect in the form of bilateral syndactyly. Digit fusion involves both soft and hard tissues, and is associated with reduced apoptosis at affected sites. Two lines of evidence suggest that syndactily is primarily due to defective mesenchyme differentiation, rather than reduced apoptosis of interdigital tissue. First, fusion occurs before appearance of interdigital cell death; second, interdigital tissues having incomplete separation fail to respond to apoptotic clues from implanted BMP-4 beads. Syndactyly is associated with a disorganized matrix, but with normal BMP gene expression. On the other hand, mice double heterozygous for null Fbn2 and Bmp7 alleles display the combined digit phenotype of both nullizygotes. Together, these results imply functional interaction between Fbn2-rich microfibrils and BMP-7 signaling. As such, they uncover an unexpected relationship between the insoluble matrix and soluble factors during limb patterning. We also demonstrate that the Fbn2- null mutation is allelic to the recessive shaker-with-syndactyly (sy) locus on chromosome 18.
Subject(s)
Body Patterning/genetics , Extracellular Matrix/metabolism , Limb Deformities, Congenital/genetics , Microfibrils/metabolism , Microfilament Proteins/deficiency , Syndactyly/genetics , Transforming Growth Factor beta , Alleles , Animals , Apoptosis , Body Patterning/drug effects , Bone Morphogenetic Protein 4 , Bone Morphogenetic Protein 7 , Bone Morphogenetic Proteins/deficiency , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Bone Morphogenetic Proteins/pharmacology , Chromosomes/genetics , Drug Implants , Fibrillin-2 , Fibrillins , Forelimb/embryology , Forelimb/pathology , Gene Targeting , Hindlimb/embryology , Hindlimb/pathology , Limb Deformities, Congenital/pathology , Mesoderm/cytology , Mice , Mice, Knockout , Microfibrils/pathology , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Signal Transduction/genetics , Syndactyly/metabolism , Syndactyly/pathologyABSTRACT
Pseudoexfoliation (PEX) syndrome is a common and clinically important systemic condition characterized by the pathologic production and accumulation of an abnormal fibrillar extracellular material in many intra- and extraocular tissues. Recent evidence suggests that it is a type of elastosis associated with the excess synthesis of elastic microfibrillar components such as fibrillin-1. Since transforming growth factor (TGF)-beta is a major modulator of extracellular matrix formation, the potential involvement of TGF-beta and its latent form binding protein (LTBP) in this aberrant matrix process was investigated. The expression of various isoforms of TGF-beta and LTBP was investigated in the anterior segment tissues of PEX and control eyes on the protein and mRNA level by light and electron microscopic immunohistochemistry, in situ hybridization, and semiquantitative RT-PCR. TGF-beta1 and TGF-beta2 levels were measured in aqueous humor and serum of PEX and control patients by ELISA. Cultures of Tenon's capsule fibroblasts were established to study the effect of TGF-beta1 on fibrillin-1 mRNA expression by Northern blot analysis. Significantly increased concentrations of both total and active TGF-beta1 were measured in the aqueous humor of PEX eyes without and with glaucoma as compared to control eyes, whereas levels of TGF-beta2 were not significantly different. The expression of TGF-beta1, LTBP-1, and LTBP-2, but not TGF-beta2, was markedly increased in anterior segment tissues of PEX eyes, particularly in the non-pigmented epithelium of the ciliary body, on both the mRNA and the protein level. Latent TGF-beta1 staining was consistently associated with PEX material deposits and could be released by proteolytic processing. Double immunolabeling revealed clear co-localization of LTBP-1 and -2 with latent TGF-beta1 and with fibrillin-1 on PEX fibrils. The expression of mRNA coding for fibrillin-1 was up-regulated in vitro by TGF-beta1. This study provides evidence for a significant role of TGF-beta1 and the LTBPs 1 and 2 in PEX syndrome. The results suggest that increased levels of latent and active TGF-beta1 in the aqueous humor of PEX patients, derived from enhanced local synthesis and activation, promote the buildup of the abnormal extracellular elastic material characteristic of PEX syndrome. They further support a dual role for LTBPs, both as integral structural components of PEX fibers and as a means of matrix anchorage of latent TGF-beta1, representing one possible mechanism for the regulation of TGF-beta1 activity in PEX eyes. Future therapeutic strategies might focus on TGF-beta1 antagonistic approaches.
Subject(s)
Carrier Proteins/physiology , Exfoliation Syndrome/metabolism , Intracellular Signaling Peptides and Proteins , Transforming Growth Factor beta/physiology , Aged , Aqueous Humor/metabolism , Blotting, Northern , Case-Control Studies , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Fibrillin-1 , Fibrillins , Fibroblasts/physiology , Humans , In Situ Hybridization/methods , Latent TGF-beta Binding Proteins , Microfilament Proteins/metabolism , Microscopy, Electron/methods , Protein Isoforms/physiology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Up-RegulationABSTRACT
Fibrillins are large structural macromolecules that are components of connective tissue microfibrils. Fibrillin microfibrils have been found in association with basement membranes, where microfibrils appear to insert directly into the lamina densa. It is unknown whether fibrillins are limited to these sites of microfibril insertion or are present throughout the lamina densa. In this study, electron microscopic immunolocalization demonstrated the presence of fibrillin-1 throughout the lamina densa in the dermal-- epidermal junction. In order to investigate whether fibrillin microfibrils might be present in the lamina densa, epithelial cell cultures (WISH, HaCaT, and primary keratinocytes) were analyzed by immunofluorescence, immunoblotting, and extraction of microfibrils followed by rotary shadowing electron microscopy and compared to mesenchymal cell cultures (dermal fibroblasts and MG63 osteosarcoma). In contrast to mesenchymal cells, which elaborate a fibrillin fibril network, epithelial cells primarily deposit fibrillin into the extracellular matrix in a nonfibrillar form. Coculture experiments using human epithelial cells and mouse fibroblasts implicated the cells themselves in the assembly of fibrillin. The importance of the cell in this process was further underscored by novel data demonstrating that keratinocytes selectively secrete fibrillin-1 into the matrix and not into the medium and can differentiate between fibrillin-1 and fibrillin-2.
Subject(s)
Dermis/cytology , Epithelial Cells/metabolism , Microfilament Proteins/metabolism , 3T3 Cells , Animals , Basement Membrane/metabolism , Cells, Cultured , Coculture Techniques , Extracellular Matrix/metabolism , Fibrillin-1 , Fibrillin-2 , Fibrillins , Fibroblasts/metabolism , Fluorescent Antibody Technique , Humans , Keratinocytes/metabolism , Mesoderm/cytology , Mice , Microfibrils/chemistry , Microfibrils/metabolism , Microfibrils/ultrastructure , Microfilament Proteins/analysis , Microscopy, ElectronABSTRACT
We report a case of vasculitis after Prosorba treatment in a patient with rheumatoid arthritis. The patient is a 66-year-old white male with long standing rheumatoid arthritis and hepatitis B. He was treated with the standard regimen for Prosorba treatment. He improved and met criteria for an American College of Rheumatology (ACR) 20% response. While on therapy he developed a nonhealing ulcer. Approximately 2 weeks after treatment was completed, he developed palpable purpura and mononeuritis multiplex. Deep dermal biopsy confirmed the presence of both small and medium vessel vasculitis. Nerve conductions studies were consistent with neuropathic conduction delays. He was treated with 1mg/kg/day of oral prednisone. Prosorba has been reported to cause leukocytoclastic vasculitis during treatment, but has not been noted to involve medium sized vessels. This patient's history and presentation are most consistent with rheumatoid arthritis associated vasculitis, though the Prosorba treatment cannot be ruled out as a cause or a contributing factor. Importantly, although Prosorba treated his synovitis, it did not prevent concomitant vasculitis.
ABSTRACT
In the last 5 years, significant progress has been made in understanding the structure and function of all the major domains composing the fibrillins. A previous review [Meth. Enzymol. 245 (1994), 29] focused on the isolation of fibrillin monomers and fibrillin-containing polymers (microfibrils). In this article, information gained from recent studies which have further elucidated molecular structure and investigated effects of mutations on structural and functional properties will be summarized. In addition, studies of functional domains in fibrillins which may be important in assembling microfibrils will be discussed. Throughout this review, the authors have attempted to identify areas of research which have been controversial. In the conclusion, we raise important questions which remain unresolved.
Subject(s)
Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Animals , Biopolymers , Calcium/metabolism , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Fibrillins , Humans , Mice , Microfibrils/metabolism , Microfilament Proteins/genetics , Mutation, Missense , Protein Structure, TertiaryABSTRACT
The Tight skin (Tsk) mutation is a duplication of the mouse fibrillin 1 (Fbn1) gene that results in a larger (418 kD) than normal (350 kD) protein; Tsk/+ mice display increased connective tissue, bone overgrowth, and lung emphysema. Lung emphysema, bone overgrowth, and vascular complications are the distinctive traits of mice with reduced Fbn1 gene expression and of Marfan syndrome (MFS) patients with heterozygous fibrillin 1 mutations. Although Tsk/+ mice produce equal amounts of the 418- and 350-kD proteins, they exhibit a relatively mild phenotype without the vascular complications that are associated with MFS patients and fibrillin 1-deficient mice. We have used genetic crosses, cell culture assays and Tsk-specific antibodies to reconcile this discrepancy and gain new insights into microfibril assembly. Mice compound heterozygous for the Tsk mutation and hypomorphic Fbn1 alleles displayed both Tsk and MFS traits. Analyses of immunoreactive fibrillin 1 microfibrils using Tsk- and species-specific antibodies revealed that the mutant cell cultures elaborate a less abundant and morphologically different meshwork than control cells. Cocultures of Tsk/Tsk fibroblasts and human WISH cells that do not assemble fibrillin 1 microfibrils, demonstrated that Tsk fibrillin 1 copolymerizes with wild-type fibrillin 1. Additionally, copolymerization of Tsk fibrillin 1 with wild-type fibrillin 1 rescues the abnormal morphology of the Tsk/Tsk aggregates. Therefore, the studies suggest that bone and lung abnormalities of Tsk/+ mice are due to copolymerization of mutant and wild-type molecules into functionally deficient microfibrils. However, vascular complications are not present in these animals because the level of functional microfibrils does not drop below the critical threshold. Indirect in vitro evidence suggests that a potential mechanism for the dominant negative effects of incorporating Tsk fibrillin 1 into microfibrils is increased proteolytic susceptibility conferred by the duplicated Tsk region.
Subject(s)
Extracellular Matrix Proteins/genetics , Extracellular Matrix/metabolism , Microfilament Proteins/genetics , Alleles , Animals , Cardiovascular Abnormalities/genetics , Crosses, Genetic , Extracellular Matrix/ultrastructure , Extracellular Matrix Proteins/ultrastructure , Fibrillin-1 , Fibrillins , Gene Duplication , Genes, Dominant , Genes, Lethal , Genotype , Heterozygote , Homozygote , Marfan Syndrome/etiology , Mice , Mice, Mutant Strains , Microfilament Proteins/ultrastructure , Phenotype , Protein Conformation , Skin Abnormalities/geneticsABSTRACT
Most extracellular proteins consist of various modules with distinct functions. Mutations in one common type, the calcium-binding epidermal growth factor-like module (cbEGF), can lead to a variety of genetic disorders. Here, we describe as a model system structural and functional consequences of two typical mutations in cbEGF modules of fibrillin-1 (N548I, E1073K), resulting in the Marfan syndrome. Large (80-120 kDa) wild-type and mutated polypeptides were recombinantly expressed in mammalian cells. Both mutations did not alter synthesis and secretion of the polypeptides into the culture medium. Electron microscopy after rotary shadowing and comparison of circular dichroism spectra exhibited minor structural differences between the wild-type and mutated forms. The mutated polypeptides were significantly more susceptible to proteolytic degradation by a variety of proteases as compared with their wild-type counterparts. Most of the sensitive cleavage sites were mapped close to the mutations, indicating local structural changes within the mutated cbEGF modules. Other cleavage sites, however, were observed at distances beyond the domain containing the mutation, suggesting longer range structural effects within tandemly repeated cbEGF modules. We suggest that proteolytic degradation of mutated fibrillin-1 may play an important role in the pathogenesis of Marfan syndrome and related disorders.
Subject(s)
Calcium/metabolism , Epidermal Growth Factor/metabolism , Marfan Syndrome/physiopathology , Microfilament Proteins/metabolism , Amino Acid Sequence , Cell Line , Electrophoresis, Polyacrylamide Gel , Fibrillin-1 , Fibrillins , Humans , Marfan Syndrome/genetics , Molecular Sequence Data , Mutation , Protein Conformation , Protein Folding , Structure-Activity RelationshipABSTRACT
Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.
Subject(s)
Cross-Linking Reagents/metabolism , Disulfides/metabolism , Microfibrils/metabolism , Microfilament Proteins/metabolism , Amino Acid Sequence , Animals , Aorta/embryology , Chick Embryo , Culture Media, Serum-Free/metabolism , Cysteine/metabolism , Electrophoresis, Polyacrylamide Gel , Extracellular Matrix Proteins/chemistry , Fibrillin-1 , Fibrillin-2 , Fibrillins , Fibroblasts/metabolism , Humans , Microfibrils/chemistry , Microfilament Proteins/chemistry , Molecular Sequence Data , Mutagenesis , Organ Culture Techniques , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Time FactorsABSTRACT
Latent transforming growth factor beta-binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor beta (TGF-beta) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67-467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF beta 1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded.
Subject(s)
Bone and Bones/metabolism , Carrier Proteins/physiology , Intracellular Signaling Peptides and Proteins , Microfibrils/metabolism , Microfilament Proteins/metabolism , Amino Acid Sequence , Blotting, Western , Bone and Bones/ultrastructure , Cell Line , Collagen/metabolism , Fibrillin-1 , Fibrillins , Fibronectins/metabolism , Fluorescent Antibody Technique , Humans , Latent TGF-beta Binding Proteins , Microscopy, Immunoelectron , Molecular Sequence DataABSTRACT
The use of alternative medicine in the United States has increased over the past 2 decades. With increasing use, the possibility of toxicity also increases. We report two cases of rhabdomyolysis related to the use of two nutritional supplements: Diet Fuel and GlutaMASS. Each of these patients was a young, healthy male who was on a steady training regimen. A review of the literature and of the U.S. Food and Drug Administration's database revealed several reports of serious adverse events associated with these supplements and their ingredients. We hypothesize that the use of the supplements combined with normal physical training activities resulted in serious muscle injury.
ABSTRACT
Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory-fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.
Subject(s)
Aortic Aneurysm/genetics , Aortic Aneurysm/pathology , Aortic Dissection/genetics , Aortic Dissection/pathology , Microfilament Proteins/genetics , Animals , Aorta/pathology , Fibrillin-1 , Fibrillins , Heterozygote , Homozygote , Kyphosis/genetics , Kyphosis/pathology , Marfan Syndrome/genetics , Mice , Mice, Knockout , Microfilament Proteins/deficiency , Microfilament Proteins/metabolism , Ribs/abnormalities , Tunica Media/pathologyABSTRACT
Fibrillin-1 is a major component of the 10 nm microfibrils of the extracellular matrix (ECM). It is synthesized as an approximately 350 kDa precursor molecule, profibrillin-1, which is proteolytically processed into its biologically active approximately 320 kDa form. Furin, a calcium-dependent endoprotease of the subtilisin family, which is known to be the processing enzyme for a variety of proproteins, is believed to be responsible for the N-terminal proteolytic cleavage of profibrillin-1. In this article we provide several lines of evidence that the C-terminal trimming of profibrillin-1 also occurs via a furin-type activity. Edman degradation of a small recombinant C-terminal subdomain of fibrillin-1 revealed complete processing of the peptide immediately after the tribasic recognition sequence (R-X-K/R-R) for furin. In vitro expression experiments using another recombinant construct consisting of the C-terminal half of fibrillin-1 indicated that disruption of the putative recognition sequence for furin by site-directed mutagenesis drastically impairs proteolytic processing of the propeptide. In addition, our results suggest that the N-terminal half of fibrillin-1 is necessary for its incorporation into the ECM.
Subject(s)
Microfilament Proteins/metabolism , Protein Precursors/metabolism , Subtilisins/metabolism , Amino Acid Sequence , Animals , COS Cells , Fibrillin-1 , Fibrillins , Furin , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Precursors/chemistry , Protein Precursors/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
PURPOSE: To characterize the expression of fibrillins, microfibril components, in human corneas with pseudophakic/aphakic (PBK/ABK) bullous keratopathy. METHODS: Normal and PBK/ABK corneas were stained by immunofluorescence for fibrillin-1 and -2. The expression of fibrillin-1 messenger RNA (mRNA) was studied by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern analysis. RESULTS: Only fibrillin-1 was detected in normal and diseased corneas. As described previously, in normal corneas, it was found in the limbal stroma and basement membrane (BM) and in the peripheral corneal epithelial BM for a short distance near the limbus. Central corneal BM, stroma, and Descemet's membrane were negative. All PBK/ABK corneas were positive for fibrillin-1, which was detected in fibrillar deposits at the endothelial face of Descemet's membrane, in the epithelial BM, subepithelial fibrosis areas, and posterior collagenous layer. By RT-PCR, low levels of fibrillin-1 mRNA were detected in normal corneas, and they increased significantly in PBK/ABK corneas. CONCLUSION: The deposition of fibrillin-1, together with tenascin-C, in PBK/ABK corneas may be part of an abnormal fibrotic/wound-healing process that occurs during the development of postsurgical corneal edema with the formation of bullae and posterior collagenous layer.