Goodpasture antigen-binding protein is a soluble exportable protein that interacts with type IV collagen. Identification of novel membrane-bound isoforms

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at least two GPBP isoforms resulting from canonical (77-kDa) and noncanonical (91-kDa) mRNA translation initiation. The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. These and previous data implicate GPBPs in a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane.     

Comparison of microCT and an inverse finite element approach for biomechanical analysis: results in a mesenchymal stem cell therapeutic system for fracture healing

An important concern in the study of fracture healing is the ability to assess mechanical integrity in response to candidate therapeutics in small-animal systems. In recent reports, it has been proposed that microCT image-derived densitometric parameters could be used as a surrogate for mechanical property assessment. Recently, we have proposed an inverse methodology that iteratively reconstructs the modulus of elasticity of the lumped soft callus/hard callus region by integrating both intrinsic mechanical property (from biomechanical testing) and geometrical information (from microCT) within an inverse finite element analysis (FEA) to define a callus quality measure. In this paper, data from a therapeutic system involving mesenchymal stem cells is analyzed within the context of comparing traditional microCT densitometric and mechanical property metrics. In addition, a novel multi-parameter regression microCT parameter is analyzed as well as our inverse FEA metric. The results demonstrate that the inverse FEA approach was the only metric to successfully detect both longitudinal and therapeutic responses. While the most promising microCT-based metrics were adequate at early healing states, they failed to track late-stage mechanical integrity. In addition, our analysis added insight to the role of MSCs by demonstrating accelerated healing and was the only metric to demonstrate therapeutic benefits at late-stage healing. In conclusion, the work presented here indicates that microCT densitometric parameters are an incomplete surrogate for mechanical integrity. Additionally, our inverse FEA approach is shown to be very sensitive and may provide a first-step towards normalizing the often challenging process of assessing mechanical integrity of healing fractures.     

Goodpasture antigen-binding protein and its spliced variant, ceramide transfer protein, have different functions in the modulation of apoptosis during zebrafish development

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the alpha3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPDelta26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpDelta26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpDelta26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.     

Goodpasture antigen-binding protein (GPBP) directs myofibril formation: identification of intracellular downstream effector 130-kDa GPBP-interacting protein (GIP130)

Goodpasture antigen-binding protein-1 (GPBP-1) is an exportable non-conventional Ser/Thr kinase that regulates glomerular basement membrane collagen organization. Here we provide evidence that GPBP-1 accumulates in the cytoplasm of differentiating mouse myoblasts prior to myosin synthesis. Myoblasts deficient in GPBP-1 display defective myofibril formation, whereas myofibrils assemble with enhanced efficiency in those overexpressing GPBP-1. We also show that GPBP-1 targets the previously unidentified GIP130 (GPBP-interacting protein of 130 kDa), which binds to myosin and promotes its myofibrillar assembly. This report reveals that GPBP-1 directs myofibril formation, an observation that expands its reported role in supramolecular organization of structural proteins to the intracellular compartment.     

A finite element inverse analysis to assess functional improvement during the fracture healing process

Assessment of the restoration of load-bearing function is the central goal in the study of fracture healing process. During the fracture healing, two critical aspects affect its analysis: (1) material properties of the callus components, and (2) the spatio-temporal architecture of the callus with respect to cartilage and new bone formation. In this study, an inverse problem methodology is used which takes into account both features and yields material property estimates that can analyze the healing changes. Six stabilized fractured mouse tibias are obtained at two time points during the most active phase of the healing process, respectively 10 days (n=3), and 14 days (n=3) after fracture. Under the same displacement conditions, the inverse procedure estimations of the callus material properties are generated and compared to other fracture healing metrics. The FEA estimated property is the only metric shown to be statistically significant (p=0.0194) in detecting the changes in the stiffness that occur during the healing time points. In addition, simulation studies regarding sensitivity to initial guess and noise are presented; as well as the influence of callus architecture on the FEA estimated material property metric. The finite element model inverse analysis developed can be used to determine the effects of genetics or therapeutic manipulations on fracture healing in rodents.     

Upper gastrointestinal haemorrhage in patients over 80 years-old

Objectives

To evaluate the patient characteristics, outcome, and prognosis of upper gastrointestinal haemorrhage in the elderly.

Material and methods

A prospective study was conducted on 103 patients aged 80 years and over, admitted to a Gastrointestinal Bleeding Unit after an episode of upper gastrointestinal bleeding. We analysed the personal history, the characteristics of the bleeding event, and whether an urgent diagnostic or therapeutic endoscopy was performed, in order to identify clinical data and endoscopic findings that may have an influence on the outcome of the haemorrhage.

Results

The major cause of the haemorrhage was peptic ulcer in 65.1%, and 60.2% of patients were on chronic treatment with non-steroidal anti-inflammatory drugs. An urgent diagnostic endoscopy was performed in all of them, identifying the source of bleeding in 94.2%, and treatment was carried out on 28.2%. The likelihood of rebleeding was 8%, and 4.9% of patients underwent emergency surgery, with an overall mortality rate of 5.8%.

Conclusions

The performance of urgent endoscopy and the application of endoscopic haemostasis are safe and effective in stopping upper gastrointestinal bleeding in the elderly. This has significantly reduced the need for emergency surgery, improving the survival of the bleeding elderly patient and preventing recurrent bleeding.     

Conformational diversity of the Goodpasture antigen, the noncollagenous-1 domain of the alpha3 chain of collagen IV

The noncollagenous-1 domain of the alpha3 chain of collagen IV networks of basement membranes is the target of an antibody-mediated inflammatory response in Goodpasture autoimmune disease. This domain when excised from basement membranes by bacterial collagenase digestion exists in two molecular forms, M(H) and M(L), that differ in cleavage site and mobility in SDS-PAGE. In the present study, M(H) and M(L) were shown to also differ with respect to epitope exposure, susceptibility to endoprotease digestion, and redox states of specific cystene residues, as determined by MS. Moreover, M(H) and M(L) assemble to form different quaternary structures, critically influencing pathogenic epitope(s) exposure and autoantibody binding. Collectively, our findings reveal that M(H) and M(L) are conformational isomers stabilized by a distinct disulfide bond connectivity, and coexist in basement membranes. The hitherto unrecognized conformational diversification of the Goodpasture autoantigen may be of relevance in pathogenesis.     

Characterization of fimbriae extracts from porcine enterotoxigenic Escherichia coli strains carrying F6 (987P) antigen.

Fimbrial extracts from porcine enterotoxigenic Escherichia coli (ETEC) strains carrying F6 (987P) intestinal colonization factor antigen wereobtained using the thermal shock method. The extracts were analyzed by SDSPAGE and immunoblotting using different fimbriae-specific antisera. Two major protein bands with molecular masses of 17.5 and 21.9 kDa were detected. The 21.9-kDa band was identified as the major subunit of F6 fimbrial antigen in strains of serogroups O9 and O141. The 17.5-kDa band was associated with porcine strains of serogroups O9 and O20.