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Previous studies have shown that energy restriction (ER) or low-fat (LF) diets have beneficial effects on high-fat (HF) diet-induced obesity and non-insulin-dependent diabetes. However, comparison between ER and low-fat diet regarding the effect on insulin resistance and lipid metabolism has not been reported. After inducing insulin resistance by HF feeding for 20 weeks, male C57BL/6J mice were divided into 3 groups. For a period of 12 weeks, group 1 received energy restriction (70% of ad libitum, HF diet), group 2 LF diet, and group 3 maintained on HF diet. Body weight and energy intake were reduced equally in ER and LF feeding. Plasma insulin levels were decreased on LF feeding, but were unchanged on ER, when compared with HF feeding. Glucose tolerance and insulin sensitivity tests revealed that insulin sensitivity was improved more efficiently by LF feeding than on ER. Plasma triglyceride (TG) levels were lower on LF feeding compared with ER and HF feeding. Measurement of hepatic very-low-density lipoprotein (VLDL)-TG production revealed a lower production after LF diet feeding or ER compared with HF diet feeding. In summary, our data show that LF diet has a higher potential than ER to improve HF diet-induced insulin resistance, and that there is an association between improvement of insulin resistance and decrease of TG levels. Copyright 2002, Elsevier Science (USA). All rights reserved.

In rheumatoid arthritis (RA), irreversible joint damage is the result of degradation of articular structures such as cartilage, bone and tendons. The plasminogen activator (PA) system has been shown to be involved in the proteolytic degradation of cartilage matrix by rheumatoid synovial fibroblasts in vitro. To study the possibilities for therapeutic intervention via inhibition of proteolysis by rheumatoid synovial fibroblasts, we compared the effects of genetherapy with three different recombinant adenovirus constructs encoding: 1) the aminoterminal fragment of urokinase (Ad.ATF), a competitive inhibitor of urokinase binding to the urokinase receptor (uPAR); 2) bovine pancreas trypsin inhibitor (Ad.BPTI), also known as Trasylol, a potent plasmin inhibitor; and 3) a hybrid protein of ATF and BPTI (Ad.ATF-BPTI), a plasmin inhibitor that can bind to the urokinase receptor. Synovial fibroblasts were isolated from RA synovial tissue. The cells were infected for 3h with either of the three virus constructs in a concentration of 10s pfu/ml and cultured on a radiolabeled cartilage matrix, produced by bovine chondrocytes cultured in alginate beads. Transfection with Ad.ATF-BPTI resulted in a decrease of matrix degradation of 40%, whereas transfection with the other virus constructs did not inhibit proteolytic activity significantly (<10%). In conclusion, cell surface uPAR bound plasmin inhibition appears to be an effective way to inhibit proteolysis by rheumatoid fibroblasts in vitro. Local inhibition of pericellular proteolysis may be an interesting target for therapeutic intervention to prevent joint destruction in RA.

Increased plasma fibrinogen levels have been identified as a risk indicator for myocardial infarction, stroke, and thrombosis. Both environmental and genetic factors make an important contribution to plasma fibrinogen levels in humans. In the present study we evaluated, in patients with serum cholesterol levels between 4 and 8 mmol/L, the relation of plasma levels and polymorphisms of fibrinogen with coronary artery disease (CAD), cross-sectionally at baseline and after a 2-year follow-up period in which they received either a placebo or pravastatin. Higher plasma fibrinogen levels (3.9 g/L) were observed at baseline in patients with the -455AA genotype than in patients with the -455GA (3.2 g/L) and -455GG (3.1 g/L) genotypes of the -455G/A fibrinogen β gene polymorphism (P < .05). Plasma levels of fibrinogen were not related to the baseline angiographic variables (mean segment diameter [MSD] and minimum obstruction diameter [MOD]), nor to the quantitative changes in these angiographic variables. However, in the placebo group, patients with the - 455AA genotype had more progression of CAD, expressed by a significantly greater decrease of the MSD and MOD, after the 2-year follow-up period than patients with the other genotypes. The - 455G/A polymorphism was related to the progression of CAD, and pravastatin therapy seemed to offset this deleterious effect. We hypothesized that the - 455A allele may promote a stronger acute-phase response in fibrinogen and that the resulting higher fibrinogen levels may form the pathogenetic basis for the stronger progression of coronary atherosclerosis. Experiments to verify this hypothesis are being proposed and advocated, in view of the possibility of identifying a genetic marker that can recognize a subgroup of patients with an increased risk who may benefit from early treatment with lipid-lowering or anticoagulant drugs.

Chemicals/CAS: cytochrome P450, 9035-51-2; retinoic acid, 302-79-4

Abstract

A specific glucagon antiserum was generated in rabbits using a derivatisation of the methionine residue in glucagon. A sensitive and specific assay for glucagon has been developed. Interference with the assay system by plasma factors was abolished by both extraction of plasma samples and the production of glucagon-free plasma for each individual. Chemicals/CAS: glucagon, 11140-85-5, 62340-29-8, 9007-92-5; Aprotinin, 9087-70-1; Arginine, 74-79-3; Glucagon, 9007-92-5; S-methylglucagon, 68600-12-4

To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the absence and the presence of ε-amino caproic acid (EACA). Occupation of the lysyl binding site in the K2 domain with EACA has a pronounced effect on the binding of FGK1K2P to a fibrin clot (C50 = 77 ± 11 nM versus 376 ± 45 nM with EACA). Deleting the lysyl binding site in the K2 domain (substitution D236N) also impairs fibrin binding but to a lesser extent (C50 = 169 ± 20 nM). Although the binding of K2P to a fibrin clot is weak (C50 = 1163 ± 490 nM), it still is 2 orders of magnitude stronger than the binding of EACA to K2P. Therefore it was surprising to find that deletion of the lysyl binding site in K2P completely abolishes fibrin binding. Even when both the F domain and the lysyl binding site were deleted, considerable fibrin binding is still observed (C50 = 557 ± 126 nM), suggesting other than F and K2- mediated interactions. The binding of FGK1K2P, FGK1K2P (D236N), GK1K2P, and GK1K2P (D236N) to fibrin could be competitively inhibited by FGK1K2P and K2P, indicating that all molecules recognize the same interaction sites on a fibrin clot. Based on these results, a new model for the interaction of t-PA with a forming fibrin clot is proposed. The fibrin binding sites in t-PA are not confined to the F and K2 domain. The main role of the lysyl binding site in the K2 domain of t-PA appears indirect rather than direct, most likely stabilizing a conformation favorable for fibrin binding.

Endothelial cells (ECs) actively regulate the extravasation of blood constituents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cells. These changes partly depend on a rise in [Ca2+](i) and activation of the Ca2+/calmodulin-dependent myosin light chain kinase. In this study, mechanisms that contribute to the thrombin-enhanced endothelial permeability were further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under the same conditions, the activity of the related protein Rac was not affected. This was accompanied by an increase in myosin light chain phosphorylation, the generation of F-actin stress fibers, and a prolonged increase in endothelial permeability. Inhibition of the RhoA target Rho kinase with the specific inhibitor Y-27632 reduced all of these effects markedly. In the presence of Y-27632, the thrombin-enhanced permeability was additionally reduced by chelation of [Ca2+](i) by BAPTA. These data indicate that RhoA/Rho kinase and Ca2+ represent 2 pathways that act on endothelial permeability. In addition, the protein tyrosine kinase inhibitor genistein reduced thrombin-induced endothelial permeability without affecting activation of RhoA by thrombin. Our data support a model of thrombin-induced endothelial permeability that is regulated by 3 cellular signal transduction pathways. Chemicals/CAS: Amides; Calcium, 7440-70-2; Enzyme Inhibitors; Hemostatics; Intracellular Signaling Peptides and Proteins; Myosin Light Chains; Protein-Serine-Threonine Kinases, EC 2.7.1.37; Protein-Tyrosine Kinases, EC 2.7.1.112; Proto-Oncogene Proteins c-akt, EC 2.7.1.37; Pyridines; Rho-associated kinase, EC 2.7.1.-; rhoA GTP-Binding Protein, EC 3.6.5.2; Thrombin, EC 3.4.21.5; Y 27632, 138381-45-0

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-ε-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7μM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction. © 2003 Elsevier Inc. All rights reserved. Chemicals / CAS: advanced glycation end product receptor, 198785-73-8, 247590-69-8; fructose, 30237-26-4, 57-48-7, 7660-25-5, 77907-44-9; glucose, 50-99-7, 84778-64-3; glyoxylic acid, 298-12-4; pentosidine, 124505-87-9; ribose, 34466-20-1, 50-69-1, 93781-19-2; Acetaldehyde, 75-07-0; advanced glycosylation end-product receptor; Amines; Arginine, 74-79-3; Fructose, 30237-26-4; Glucose, 50-99-7; glycolaldehyde, 141-46-8; Glycosylation End Products, Advanced; Glyoxylates; glyoxylic acid, 298-12-4; Ligands; Lysine, 56-87-1; Membrane Proteins; N(6)-carboxymethyllysine, 5746-04-3; pentosidine, 124505-87-9; Receptors, Immunologic; Recombinant Proteins; Ribose, 50-69-1; Serum Albumin, Bovine Molecular Sequence Numbers: GENBANK: CAA76847;

Objective. Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular cartilage. Methods. To increase AGE levels, human adult articular cartilage was incubated with threose. The stiffness of the collagen network was measured as the instantaneous deformation (ID) of the cartilage and as the change in tensile stress in the collagen network as a function of hydration (osmotic stress technique). AGE levels in the collagen network were determined as: Nε-(carboxy[m]ethyl)lysine, pentosidine, amino acid modification (loss of arginine and [hydroxy-]lysine), AGE fluorescence (360/460 nm), and digestibility by bacterial collagenase. Results. Incubation of cartilage with threose resulted in a dose-dependent increase in AGEs and a concomitant decrease in ID (r = -0.81, P < 0.001; up to a 40% decrease at 200 mM threose), i.e., increased stiffness, which was confirmed by results from the osmotic stress technique. The decreased ID strongly correlated with AGE levels (e.g., AGE fluorescence r = -0.81, P < 0.0001). Coincubation with arginine or lysine (glycation inhibitors) attenuated the threose-induced decrease in ID (P < 0.05). Conclusion. Increasing cartilage AGE crosslinking by in vitro incubation with threose resulted in increased stiffness of the collagen network. Increased stiffness by AGE crosslinking may contribute to the age-related failure of the collagen network in human articular cartilage to resist damage. Thus, the age-related accumulation of AGE crosslinks presents a putative molecular mechanism whereby age is a predisposing factor for the development of OA.

Subchondral bone provides structural support to the overlying articular cartilage, and plays an important role in osteochondral diseases. There is growing insight that the mechanical features of bone are related to the biochemistry of the collagen network and the mineral content. In the present study, part of the normal developmental process and the influence of physical activity on biochemical composition of subchondral bone was studied. Water content, calcium content and characteristics of the collagen network (collagen, hydroxylysine, lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) crosslinking) of subchondral bone were measured in newborn foals, 5-month-old foals (pasture-grown and box-confined) and 11-month-old foals at 2 differently loaded sites of the proximal articular surface of the first phalanx. During the first 5 months postpartum, water and hydroxylysine content decreased significantly while calcium and collagen content and the amount of HP and LP crosslinks increased significantly. The withholding of physical activity during this developmental phase affected the biochemical characteristics of subchondral bone only at the site that is loaded during physical exercise. At this site, calcium content and both HP and LP crosslink levels increased significantly less than in pasture-raised animals. During development from 5-11 months, measured parameters remained essentially constant, except for water content, which decreased further. It is concluded that substantial changes, presumed to be largely exercise-driven, take place during the normal process of development in the biochemical composition of equine subchondral bone. Normal development of subchondral bone is presumably important for the normal functional adaptation of this bone to the loading conditions it is subjected to and therefore essential to resist the future biomechanical challenges the horse will encounter during its athletic career. The findings from this study and the assumed important role of subchondral bone quality in the pathogenesis of osteochondral disease merit more attention to the role of the collagen network in subchondral bone.

The objective of this study was to document the development of biochemical heterogeneity from birth to maturity in equine articular cartilage, and to test the hypothesis that the amount of exercise during early life may influence this process. Neonatal foals showed no biochemical heterogeneity whatsoever, in contrast to a clear biochemical heterogeneity in mature horses. The process of formation of site differences was almost completed in exercised foals age 5 months, but was delayed in those deprived of exercise. For some collagen-related parameters, this delay was not compensated for after an additional 6 month period of moderate exercise. It is concluded that the functional adaptation of articular cartilage, as reflected in the formation of biochemical heterogeneity in the horse, occurs for the most part during the first 5 months postpartum. A certain level of exercise seems essential for this process and withholding exercise in early life, may result in a delay in the adaptation of the cartilage.

Objective. To investigate the effects of doxycycline on disease activity and joint destruction in patients with rheumatoid arthritis (RA). Methods. A 36 week double blind, placebo controlled crossover trial was conducted. Patients (n = 66) received 50 mg doxycycline or placebo twice a day during 12, 24, or 36 weeks. Patient assessments were performed before the treatment was administered, at 6, 12, 24 and 36 weeks of treatment, and finally at 4 weeks after cessation of treatment. Patient assessments, swollen and tender joint counts, duration of morning stiffness, erythrocyte sedimentation rate, and Modified Disease Activity Score were used as measures of disease activity. Effects on joint destruction were assessed by urinary excretion of the pyridinolines hydroxylysylpyridinoline and lysylpyridinoline and by scoring radiographic damage of hands and feet before and after treatment. Results. The changes of clinical or laboratory disease activity measures, pyridinoline excretion, or progression of radiographic joint damage during doxycycline or placebo treatment did not differ significantly. Conclusion. The results indicate that 50 mg doxycycline twice a day provided no therapeutic benefit for patients with RA.