Long term evaluation of disease progression through the quantitative magnetic resonance imaging of symptomatic knee osteoarthritis patients: correlation with clinical symptoms and radiographic changes

The objective of this study was to further explore the cartilage volume changes in knee osteoarthritis (OA) over time using quantitative magnetic resonance imaging (qMRI). These were correlated with demographic, clinical, and radiological data to better identify the disease risk features. We selected 107 patients from a large trial (n = 1,232) evaluating the effect of a bisphosphonate on OA knees. The MRI acquisitions of the knee were done at baseline, 12, and 24 months. Cartilage volume from the global, medial, and lateral compartments was quantified. The changes were contrasted with clinical data and other MRI anatomical features. Knee OA cartilage volume losses were statistically significant compared to baseline values: -3.7 ± 3.0% for global cartilage and -5.5 ± 4.3% for the medial compartment at 12 months, and -5.7 ± 4.4% and -8.3 ± 6.5%, respectively, at 24 months. Three different populations were identified according to cartilage volume loss: fast (n = 11; -13.2%), intermediate (n = 48; -7.2%), and slow (n = 48; -2.3%) progressors. The predictors of fast progressors were the presence of severe meniscal extrusion (p = 0.001), severe medial tear (p = 0.005), medial and/or lateral bone edema (p = 0.03), high body mass index (p < 0.05, fast versus slow), weight (p < 0.05, fast versus slow) and age (p < 0.05 fast versus slow). The loss of cartilage volume was also slightly associated with less knee pain. No association was found with other Western Ontario McMaster Osteoarthritis Index (WOMAC) scores, joint space width, or urine biomarker levels. Meniscal damage and bone edema are closely associated with more cartilage volume loss. These data confirm the significant advantage of qMRI for reliably measuring knee structural changes at as early as 12 months, and for identifying risk factors associated with OA progression.     

The accumulation of triacylglycerols within the endoplasmic reticulum of developing seeds of Helianthus annuus

Microsomes isolated from developing seeds of Helianthus annuus were prepared in a medium which ensured that endoplasmic reticulum (ER)-bound polysomes remained attached to the ER during homogenization. The microsomes were then incubated with the substrates necessary to sustain the synthesis of triacylglycerols (TAGs). Microsomes that contained high activities of the enzymes involved in the synthesis of TAGs (the enzymes of the Kennedy pathway) accumulated TAGs synthesized in vitro , resulting in a decrease in their buoyant density. These light membrane fractions could therefore be separated on discontinuous sucrose density gradients from microsomes containing low activities of the enzymes of the Kennedy pathway. Analysis of the microsome fractions by ¹H-NMR spectroscopy showed that the TAGs synthesized in the microsomes in vitro were tumbling isotropically in an environment similar to that of the TAGs in oil bodies. Western blot analysis revealed that microsomes which synthesized large amounts of TAGs in vitro were also substantially enriched in oleosins. In addition, labelling studies indicated that the oleosins newly synthesized in vitro by ‘run-on' translation of ER-bound polysomes also localized to light membrane fractions. This indicates that oleosins are specifically enriched in regions of the ER involved in the biogenesis of the oil body.     

Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1.

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.     

Evidence that amylase is released from two distinct pools of secretory proteins in the pancreas

Previous experiments demonstrated the existence of at least two pools of secretory proteins in the exocrine pancreas. We have measured the specific activities of amylase released under resting conditions and of amylase in the zymogen granules. Specific activity of resting secretion was twice that found under stimulated conditions or in zymogen granules. Secretory proteins were pulse-labeled and amylase was measured after precipitation of the enzyme with glycogen. Pancreatic juice collected at 45-50 min post-pulse contained 10-25-times the amylase activity found in zymogen granules. These results confirm the existence of at least two distinct pools of secretory proteins in the exocrine pancreas and suggest the existence of an intracellular route of secretory proteins which would bypass the zymogen granule compartment.     

An Arabidopsis immunophilin, AtFKBP12, binds to AtFIP37 (FKBP interacting protein) in an interaction that is disrupted by FK506

AtFKBP12 is an Arabidopsis cDNA that encodes a protein similar to the mammalian immunophilin, FKBP12. AtFKBP12 was used as ‘bait’ in a yeast 2-hybrid system to screen for cDNAs in Arabidopsis encoding proteins that bind to FKBP12. Two partial cDNAs were recovered encoding the C-terminus of a protein we have called Arabidopsis thaliana FKBP12 interacting protein 37 (AtFIP37). AtFIP37 is similar to a mammalian protein, FAP48, that also binds to FKBP12. The interaction between AtFKBP12 and AtFIP37 in the 2-hybrid system, as assessed by histidine auxotrophy and β-galactosidase activity, was disrupted by FK506, but not by cyclosporin A, a drug that binds to cyclophilin A. AtFIP37 was also shown to bind in vitro to AtFKBP12 in GST-fusion protein binding assays. The binding was abolished by prior incubation of AtFKBP12 with FK506. These findings indicate that an Arabidopsis FKBP12 ortholog encodes a protein that binds FK506 and that the interaction between AtFKBP12 and AtFIP37 may involve the FK506 binding site of AtFKBP12. The interaction provides interesting new opportunities for controlling protein:protein interactions in vivo in plants.     

Newly synthesized secretory proteins from pig pancreas are not released from a homogeneous granule compartment

The pancreatic secretion of anesthetized pigs was collected by cannulation after pulse labeling with [³H]leucine. Collection at 5 min intervals started immediately post-pulse labeling up to 85 min. The volume, the protein content and the trichloroacetic acid-precipitable radioactivity of the juice were measued. The specific radioactivity of the secertory proteins was compared to that of a zymogen granule fraction isolated from the same animal. The latter was very much higher. Caerulein stimulation for 5 min at 80 min post-pulse caused a sharp drop in the specific activity of secretory proteins in the juice, to a level lower than that of the zymogen granule content. These data support the concept of more than one pool of secretory proteins in the pancreas and are incompatible with the concept that secretory proteins derive from an homogeneous granule compartment in a functionally homogeneous population of cells. To explain our results the hypothesis of a second intracellular route for the secretory proteins is proposed.     

A unique secretory behavior for GP2 in the exocrine pancreas.

GP2 is the major glycoprotein component of the zymogen granule membrane of pancreas acinar cell. It was recently found that this protein is secreted and forms a network of fibrils in pancreatic juice. In the present work, with an ELISA, we have examined the fluctuations of GP2 levels in the juice collected in resting and stimulated conditions. In anaesthetized fasting rats, GP2 represented about 6-8% of total protein output. Stimulation by caerulein, carbachol, or their combination caused an immediate and significant burst in both protein and GP2 outputs. However, the GP2 increase did not parallel protein release. Its relative proportions decreased with the intensity of the stimulus. The secretory behavior of GP2 cannot be explained with the current concepts of constitutive and regulated pathways of secretion and suggests the existence of a yet undefined mode of secretion for this protein.     

Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings [12]. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the -glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.     

Antiserum Inactivation of Electrophoretically Purified M13 Diploid Virions: Model for the F-Specific Filamentous Bacteriophages

Antiserum inactivation experiments were carried out on electrophoretically purified diploid virions from a cross between two complementing amber mutants of phage M13. The total (homozygous plus heterozygous) diploid population, assayed on a permissive host where only one genome is needed for plaque formation, was inactivated at the same rate as haploids. Heterozygous diploids, assayed on a nonpermissive host, where both genomes are needed for plaque formation, were twice as sensitive as haploids and the total diploid population. These results have led us to propose a model for serum inactivation of the F-specific filamentous phages. According to this model, phage-neutralizing antibodies attach anywhere along the length of the phage and allow the phage to penetrate only up to the first bound antibody molecule.     

Small-Angle x-ray scattering and crystallographic studies of arcelin-1: An insecticidal lectin-like glycoprotein from Phaseolus vulgaris L

Arcelin-1 and α-amylase inhibitor are two lectin-like glycoproteins expressed in the seeds of the kidney bean (Phaseolus vulgaris). They display insecticidal activities and protect the seeds from predation by larvae of various bruchids through different biological actions. Solution-state investigations by small-angle X-ray scattering (SAXS) show the dimeric structure of arcelin-1, a requirement for its hemagglutinating properties. Anions were found to have specific properties in their effectiveness to disrupt protein aggregates, affect solubility, and improve crystallizability. The SAXS results were used to improve crystallization conditions, and single crystals diffracting beyond 1.9 Å resolution were obtained. X-ray diffraction data analysis shows that noncrystallographic symmetry-related arcelin-1 molecules form a lectin-like dimer and reveals the presence of a solvent-exposed anion binding site on the protein, at a crystal-packing interface. The solution state properties of arcelin-1 and crystal twinning may be explained by the anion specificity of this binding site. Proteins 29:433–442, 1997. © 1997 Wiley-Liss, Inc.