The experience of brace treatment in children/adolescents with scoliosis

Background

In a previous study, a number of genes, associated with spine musculoskeletal deformity phenotypes in mouse and in synteny between mouse and man, were identified as candidate genes for IS. Among these genes, MATN 1, which carries a polymorphic microsatellite marker within its sequence, was selected for a linkage analysis. MATN1 is localised at 1p35 and is mainly expressed in cartilage. The objective of this study was to assess a linkage disequilibrium between the matrilin-1 (MATN 1) gene and the idiopathic scoliosis (IS).

Methods

The genetic study was conducted on a population of 81 trios, each consistent of a daughter/son affected by idiopathic scoliosis (IS) and both parents. In all trios components, the region of MATN1 gene containing the microsatellite marker was amplified by a polymerase chain reaction. The amplicons were analysed by a DNA sequencer-genotyper. The statistical linkage analysis was performed using the extended transmission/disequilibrium test.

Results

Three microsatellite polymorphisms, respectively consisting of 103 bp, 101 bp and 99 bp, were identified. ETDT evidenced a significant preferential transmission for the 103 bp allele (Chi-square = 5.058, df = 1, P = 0.024)

Conclusion

The results suggest that the familial idiopathic scoliosis is associated to the MATN1 gene.     

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression

Introduction

Deletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra).

Methods

The effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA.

Results

rhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA.

Conclusion

Our findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.     

Identification of G proteins mediating fungal elicitor-induced dephosphorylation of host plasma membrane H+ -ATPase

Tomato cells (with the Cf-5 resistance gene) were treated withelicitor preparations containing the avr5 gene product fromtwo Cf-5 incompatible races of the fungal pathogen Cladosporiumfulvum (race 2.3 and race 4), or with elicitor preparationscontaining no avr5 gene product from two Cf-5 compatible races(race 5 and race 2.4.5.9 [EC] .11). Elicitor preparations from race2.3 or race 4 caused dephosphorylation of host plasma membraneH⁺ -ATPase in isolated plasma membranes, while the preparationsfrom race 5 or race 2.4.5.9 [EC] .11 did not. GTP()S, AlF₄^–and cholera toxin (CTX) each induced similar dephosphorylationin the absence of active elicitors. The elicitor-induced dephosphorylationof the H⁺ -ATPase was blocked by preincubation of membraneswith an antibody raised against a stimulatory G protein -subunit(anti-G_s This antibody cross-reacted with a 42 kDa polypeptidefrom tomato plasma membranes. A 42 kDa polypeptide was alsoADP-ribosylated by CTX. When plasma membranes were treated withelicitor preparations from race 4 and separated on non-dissociatingPAGE, two proteins were detected on Western blots with the antibodyraised against the -subunit, suggesting the dissociation ofthe trimeric complex. No dissociation of the complex was detectedwith antibodies raised against either the - or ß-subunitswhen the plasma membranes were treated with elicitor preparationsfrom race 5. The results provide evidence for the activationof a stimulatory subtype of trimeric G proteins in the stimulationof elicitor-induced host defences to fungal pathogens. Key words: G protein, dephosphorylation, H⁺ -ATPase, fungal elicitor, tomato     

LAPTM4B Allele *2 Is a Marker of Poor Prognosis for Gallbladder Carcinoma

Background

Lysosomal protein transmembrane 4 beta (LAPTM4B) is a novel cancer-related gene which has two alleles designated LAPTM4B*1 and LAPTM4B*2. In this study we investigated the correlation of LAPTM4B genotype with prognosis and clinicopathologic features in patients who had undergone curative resection for gallbladder carcinoma (GBC).

Methodology/Principal Findings

PCR assay was performed to determine the LAPTM4B genotype in 85 patients. The correlation of LAPTM4B genotype with clinicopathologic parameters was assessed with the Chi-squared test. Differences in patient survival were determined by the Kaplan–Meier method. Multivariate analysis of prognostic factors was carried out with Cox regression analysis. Patients with LAPTM4B *2 had both significantly shorter overall survival (OS) and shorter disease-free survival (DFS) (both P<0.001). Multivariate analysis showed that LAPTM4B genotype is a prognostic factor for OS and DFS (both P<0.001).

Conclusions/Significance

LAPTM4B allele *2 is a risk factor associated with poor prognosis in patients with resected GBC, and LAPTM4B status may be therefore be useful preoperatively as an adjunct in evaluation of the operability of GBC.     

RNA Polymerase Activity During Breakage of Seed Dormancy by Low Temperature Treatment of Fruits of Acer platanoides (Norway Maple)

Slater, R. J. and Bryant, J. A. 1987. RNA polymerase activityduring breakage of seed dormancy by low temperature treatmentof fruits of Acer platanoides (Norway maple).—J. exp.Bot. 38:1026–1032. Endogenous RNA polymerase activity has been characterized innuclei isolated from embryo axes of Acer platanoides. Optimalactivity was recorded at 4·0 mol m^–3 MgCl₂ and50 mol m^–3 (NH₄)₂SO₄ and total activity could be inhibitedby up to 30% by -amanitin. Stratification of fruits leads toa stimulation of RNA polymerase activity. A minimum of 3 d coldtreatment is required with at least 3-fold stimulation recordedafter 10 d at 4°C. The increased enzyme activity is resistantto -amanitin suggesting an effect on RNA polymerase I. Key words: Acer platanoides, RNA polymerase, seed dormancy     

Primary structure of human matrilin-2, chromosome location of the MATN2 gene and conservation of an AT-AC intron in matrilin genes

We isolated full-length cDNA clones for human matrilin-2, an oligomeric protein, which forms filamentous networks in the extracellular matrices of various tissues. The human matrilin-2 precursor is encoded by a 4.0-kb mRNA, it consists of 956 amino acids and shows 93% similarity to the mouse protein. Out of the two von Willebrand factor type A-like domains, the 10 epidermal growth factor-type modules, one unique sequence and the oligomerization module, the first A domain is the most conserved. RT-PCR demonstrated wide expression of the gene in human cell lines of fibroblastic or epithelial origin. Alternative splicing affected only 19 amino acids in a 75-moiety-long segment, unique to matrilin-2. Isolation and analysis of the 3' end of the gene revealed that the reason for alternative splicing is alternative 3' splice site selection. Further, we identified in the human matrilin-2 gene a U12 type AT-AC intron between the last two exons encoding the oligomerization domain. We mapped the matrilin-2 gene (MATN2) by fluorescence in situ hybridization at chromosome position 8q22.     

LAPTM4B allele *2 is a marker of poor prognosis following hepatic tumor resection for hepatocellular carcinoma

Background

Lysosomal protein transmembrane 4 beta (LAPTM4B) is a gene related to hepatocellular carcinoma that has two alleles designated LAPTM4B*1 and LAPTM4B*2. This study aimed to investigate the correlation of LAPTM4B genotype with prognosis and clinicopathologic features in patients who have undergone resection for hepatocellular carcinoma (HCC).

Methodology/Principal Findings

The LAPTM4B genotype was analyzed by PCR in 68 patients who had undergone curative hepatic resection for hepatocellular carcinoma. The correlation of LAPTM4B genotype with clinicopathologic parameters was assessed with the Chi-squared test. Differences in patient survival were determined by the Kaplan–Meier method. Multivariate analysis of prognostic factors was carried out with Cox regression analysis. Patients with LAPTM4B *2 had both significantly shorter overall survival (OS) and shorter disease-free survival (DFS) (both P<0.001). Multivariate analysis showed that LAPTM4B genotype is an independent prognostic factor for OS and DFS (both P<0.001).

Conclusions/Significance

Allele *2 of LAPTM4B is a risk factor associated with poor prognosis in patients with resected HCC. LAPTM4B status may be useful preoperatively as an adjunct in evaluation of the operability of HCC.     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

srb: a Bacillus subtilis Gene Encoding a Homologue of the {alpha}-Subunit of the Mammalian Signal Recognition Particle Receptor

We cloned a Bacillus subtilis gene (srb) encoding a homologueof the mammalian signal recognition particle receptor -subunit(SR). The gene is 987 bp in length and encodes a 329-amino acidprotein. The deduced amino acid sequence of the protein shared26.6, 36.2 and 49.7% identity with those of mammalian SR, archaebacterialDP and Escherichia coli FtsY, respectively. The protein containsthree conserved GTP-binding elements like the other three SRPreceptor proteins, though the N-terminal portion of the putativeB. subtilis protein was shorter than the others. Secondary structureprediction showed that an amphipathic -helix is positioned inthe N-terminal region. A defect in srb inhibited cell growthand protein translocation.     

A role for the ubiquitin ligase Nedd4 in membrane sorting of LAPTM4 proteins

Background

The Lysosome associated protein transmembrane (LAPTM) family is comprised of three members: LAPTM5, LAPTM4a and LAPTM4b, with the latter previously shown to be overexpressed in numerous cancers. While we had demonstrated earlier the requirement of the E3 ubiquitin ligase Nedd4 for LAPTM5 sorting to lysosomes, the regulation of sorting of LAPTM4 proteins is less clear.

Methodology/Principal Findings

Here we show that LAPTM4a and LAPTM4b are localized to the lysosome, but unique to LAPTM4b, a fraction of it is present at the plasma membrane and its overexpression induces the formation of actin-based membrane protrusions. We demonstrate that LAPTM4s, like LAPTM5, are able to co-immunoprecipitate with the E3 ubiquitin ligase Nedd4, an interaction that is dependent on LAPTM4 PY motifs and plays a role in membrane sorting. Accordingly, in Nedd4 knockout mouse embryonic fibroblasts (MEFs), LAPTM4a and LAPTM4b show reduced lysosomal localization. Moreover, lack of PY motifs leads to enhanced missorting of LAPTM4b to the plasma membrane instead of the lysosome.

Conclusions/Significance

These results suggest that while some requisites of LAPTM5 lysosomal sorting are conserved among LAPTM4 proteins, LAPTM4a and LAPTM4b have also developed distinct sorting requirements.