Detection of quantitative trait loci for backfat thickness and intramuscular fat content in pigs (Sus scrofa).

In an experimental cross between Meishan and Dutch Large White and Landrace lines, 619 F(2) animals and their parents were typed for molecular markers covering the entire porcine genome. Associations were studied between these markers and two fatness traits: intramuscular fat content and backfat thickness. Association analyses were performed using interval mapping by regression under two genetic models: (1) an outbred line-cross model where the founder lines were assumed to be fixed for different QTL alleles; and (2) a half-sib model where a unique allele substitution effect was fitted within each of the 19 half-sib families. Both approaches revealed for backfat thickness a highly significant QTL on chromosome 7 and suggestive evidence for a QTL at chromosome 2. Furthermore, suggestive QTL affecting backfat thickness were detected on chromosomes 1 and 6 under the line-cross model. For intramuscular fat content the line-cross approach showed suggestive evidence for QTL on chromosomes 2, 4, and 6, whereas the half-sib analysis showed suggestive linkage for chromosomes 4 and 7. The nature of the QTL effects and assumptions underlying both models could explain discrepancies between the findings under the two models. It is concluded that both approaches can complement each other in the analysis of data from outbred line crosses.

Alignment of the PiGMaP and USDA linkage maps of porcine chromosomes 3 and 9.

The European Pig Gene Mapping Project (PiGMaP) and United States Department of Agriculture (USDA) porcine linkage maps for chromosomes 3 and 9 have been aligned by typing three USDA microsatellites from chromosome 3 and five from chromosome 9 on the PiGMaP reference families. Using the CRIMAP linkage analysis package, revised multipoint linkage maps were constructed for chromosome 3 and 9. Inclusion of these USDA markers in the multipoint analysis resulted in an increase in length of 47% and 33% respectively for these two PiGMaP linkage groups. This increase in size is mainly the result of extension of the ends of both linkage groups.

Expression of Escherichia coli branching enzyme in tubers of amylose-free transgenic potato leads to an increased branching degree of the amylopectin.

In order to increase the branching degree of potato tuber starch, the gene encoding branching enzyme (glgB) of Escherichia coli was expressed in the amylose-free potato mutant. The E. coli glgB was cloned in the binary vector pBIN19 under the transcriptional control of the potato Granule Bound Starch Synthase (GBSS) promoter and transitpeptide sequence. The E. coli glgB was cloned behind the two N-terminal amino acids of the GBSS mature protein, creating a chimeric protein. Transgenic plants were obtained which expressed the E. coli branching enzyme as was shown by the presence of mRNA and protein in the tubers. Correctly processed protein was found both in the soluble and starch granule bound protein fraction. Analysis of the starch showed an increase in the branching degree (DE) of up to 25% more branchpoints. The increase in the number of branchpoints was due to the presence of more short chains, with a degree of polymerization (DP) of 16 glucose-residues or less in the amylopectin. Changes in other characteristics of the starch, such as average chain length (CL) and lambda max, indicated a more branched structure for starch of transformed plants as well.

Expression of a wild-type GBSS gene introduced into an amylose-free potato mutant by Agrobacterium tumefaciens and the inheritance of the inserts at the microsporic level.

Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Transformation of a diploid amylose-free (amf) potato mutant with the gene encoding GBSS leads to the restoration of amylose synthesis. Transformants were obtained which had wild-type levels of both GBSS activity and amylose content. It proved to be difficult to increase the amylose content above that of the wild-type potato by the introduction of additional copies of the wild-type GBSS gene. Staining of starch with iodine was suitable for investigating the degree of expression of the inserted GBSS gene in transgenic amf plants. Of the 19 investigated transformants, four had only red-staining starch in tubers indicating that no complementation of the amf mutation had occured. Fifteen complemented transformants had only blue-staining starch in tubers or tubers of different staining categories (blue, mixed and red), caused either by full or partial expression of the inserted gene. Complementation was also found in the microspores. The segregation of blue- and red-staining microspores was used to analyse the inheritance of the introduced GBSS genes. A comparison of the results from microspore staining and Southern hybridisation indicated that, in three tetraploid transgenics, the gene was probably inserted before (duplex), and in all others after, chromosome doubling (simplex). The partial complementation was not due to methylation of the HPAII/MSPI site in the promoter region. Partially complemented plants had low levels of mRNA as was found when the GBSS expression levels were inhibited by anti-sense technology.

High susceptibility of human articular cartilage glycosaminoglycan synthesis to changes in inorganic sulfate availability.

The effect of sulfate concentration in the medium on glycosaminoglycan synthesis in articular cartilage of five different species was examined in relation to the physiological serum sulfate concentration in these species. Only the rate of sulfated glycosaminoglycan synthesis in human articular cartilage was sensitive to small deviations from the physiological sulfate concentration. A reduction in the sulfate concentration from 0.3 mM (physiological) to 0.2 mM resulted in a 33% reduction in glycosaminoglycan synthesis. In addition, we studied the effect of arthritic and "osteoarthritic" alterations in murine cartilage on the dependence of glycosaminoglycan synthesis on low sulfate concentrations. Arthritic and "osteoarthritic" cartilage had a similar dependence on the sulfate concentration in the medium as normal cartilage. Glycosaminoglycan synthesis in human articular cartilage appears to be very sensitive to the potential sulfate-depleting effects of drugs used in the treatment of rheumatoid arthritis and osteoarthritis.

The effect of chronic paracetamol administration to rats on the glycosaminoglycan content of patellar cartilage.

Male Wistar rats were treated with paracetamol (200 mg/kg twice a day) for 2, 3, 4 and 9 weeks. During the first four weeks of paracetamol administration the serum sulfate concentration was significantly decreased. However, during the fourth until the ninth week, the serum sulfate concentration was only diminished to a small and insignificant extent. The paracetamol administration did not lead to serious liver or renal toxicity, as determined by alanine aminotransferase and creatinine levels in the serum of the rats. The paracetamol-induced serum sulfate depletion, observed during the first four weeks of the experiment, led to a significantly lower glycosaminoglycan content of the patellar cartilage of the rats after three and four weeks paracetamol treatment. When after the fourth week the serum sulfate concentration rose to nearly normal levels also the glycosaminoglycan content in the rat patellar cartilage reached control levels. These data indicate that the serum sulfate depletion might be the causative factor for the observed reduction in glycosaminoglycan content of rat patellar cartilage.

Impact of NSAIDS on murine antigen induced arthritis. II. A light microscopic investigation of antiinflammatory and bone protective effects.

The impact of daily, orally administered nonsteroidal antiinflammatory drugs (NSAID) or a steroid on arthritic knee joints of mice with 1-3 weeks of antigen induced arthritis (AIA) was examined by light microscopy. Prophylactic drug effects on inflammatory cell infiltrate in synovium and joint cavity were evident only in mice treated with prednisolone (0.2-5.0 mg/kg). Treatment with diclofenac, piroxicam (both 0.5-7.5 mg/kg) or tiaprofenic acid (3-30 mg/kg) did not change these phlogistic features significantly. Striking, however, was the protective effect of piroxicam on bone apposition, a common trait in murine AIA. Among the other drugs, minor antiosteophyte effects were exerted only by prednisolone. Histological assessment was also performed on osteocyte death which was almost exclusively confined to menisci and tibial subchondral bone. Because of variability in occurrence and intensity, no clear picture emerged from drug effects on this lethal feature. Combined data from this and a previous pharmacological study, directed at chondroprotective effects, points to prednisolone as the best antiinflammatory and joint protective drug.

Decrease of inorganic blood sulfate following treatment with selected antirheumatic drugs: potential consequence for articular cartilage.

The elimination kinetics of inorganic blood sulfate in mice was followed for four hours after a single, oral administration of an antirheumatic drug. Sodium salicylate, aspirin, diflunisal and benorylate, all in a dose of 1.25 mmol/kg, reduced the sulfate level to the less than half that of control. This phenomenon was also demonstrated by phenylbutazone, oxyphenbutazone (both 1 mmol/kg), chloroquine diphosphate (0.6 mmol/kg) and tiaprofenic acid (0.02-0.35 mmol/kg). Niflumic acid (1.08 mmol/kg), piroxicam (0.03 mmol/kg), indomethacin (6.10(-3) mmol/kg), diclofenac (5.10(-3) mmol/kg), ketoprofen (0.2 mmol/kg), naproxen (0.08 mmol/kg) and ibuprofen (0.24 mmol/kg) possessed no sulfate lowering properties. The potential relevance of the use of sulfate lowering drugs for articular cartilage integrity is discussed in the light of what is already known about this subject.

Impact of NSAIDS on murine antigen induced arthritis. I. Investigation of antiinflammatory and chondroprotective effects.

The well known nonsteroidal antiinflammatory drugs (NSAID) diclofenac, piroxicam and tiaprofenic acid as well as the corticosteroid prednisolone, were examined for their effect on injury to knee joint structures evoked by a developing murine antigen induced arthritis. To ensure the human therapeutic dose, each drug was given twice a day per os in 3 different dosage regimes. The severity of arthritis was monitored with 99mTc-uptake measurements. After 3 weeks, mice were sacrificed and knee joints were examined histologically. All drugs exhibited, in comparable extent, antiinflammatory effects in terms of swelling. Grading of joint damage was focused on cartilage harm and chondrocyte activity. Only prednisolone provided sufficient protection and seemed to stimulate repair activity. The NSAID had no clear beneficial or adverse influences on affected cartilage. Apparently, under these experimental circumstances, only prednisolone functions chondroprotectively.

The effect of low sulfate concentrations on the glycosaminoglycan synthesis in anatomically intact articular cartilage of the mouse.

We have studied the effect of environmental sulfate concentration on the glycosaminoglycan synthesis of anatomically intact patellar cartilage of the mouse in vitro. Incubation of mouse patellae in medium with sulfate concentrations below 0.5 mM resulted in a diminished incorporation of sulfate but in unaltered incorporation of glucosamine. This suggested the synthesis of undersulfated glycosaminoglycans under these conditions. We characterized glycosaminoglycans synthesized at three different sulfate concentrations: a sulfate concentration physiological for the mouse (1.0 mM), a sulfate concentration in the range where sulfate incorporation was strongly diminished (0.1 mM), and an extremely low sulfate concentration (10 nM). Analysis of glycosaminoglycan disaccharides and DEAE anion chromatography of the glycosaminoglycans could not confirm the synthesis of undersulfated glycosaminoglycans at 0.1 mM. The chromatogram of glycosaminoglycans synthesized in medium containing 10 nM showed the presence of a very low sulfated glycosaminoglycan pool not observed at higher medium sulfate concentrations. Intermediately sulfated glycosaminoglycans were also synthesized during incubation with 10 nM sulfate. So, our data indicate that only very low sulfate concentrations in the medium lead to the synthesis of undersulfated glycosaminoglycans and that the sulfation mechanism of murine patellar cartilage chondrocytes does not seem to fit completely in an "all-or-nothing" pattern.

Inhibition of glycosaminoglycan synthesis in anatomically intact rat patellar cartilage by paracetamol-induced serum sulfate depletion.

We have studied the effect of low sulfate concentrations on the glycosaminoglycan synthesis in rat patellar cartilage in vivo as well as in vitro. The oral administration of 200 mg/kg paracetamol to male Wistar rats resulted in a significant reduction of the serum sulfate concentration. Reduced serum sulfate availability resulted in a 34% decrease of glycosaminoglycan synthesis in patellar cartilage. This is due to sulfate depletion since paracetamol had no direct effects on glycosaminoglycan synthesis and a slight but significant inhibitory effect on the catabolism of radiolabeled glycosaminoglycans in vitro. The glycosaminoglycans synthesized at low sulfate concentrations in vivo were similar to the glycosaminoglycans synthesized at physiological sulfate concentrations. Studying the effect of sulfate availability in vitro on glycosaminoglycan synthesis in patellar cartilage we found that incubation of rat patellae in medium containing less than 0.5 mM inorganic sulfate led to a decreased sulfate incorporation. The use of potential sulfate decreasing drugs can lead to inhibition of glycosaminoglycan synthesis. This argues for a reconsideration of the use of these drugs in patients with already dysfunctioning cartilage metabolism as in rheumatoid arthritis and osteoarthrosis.

Synthesis of aberrant glycosaminoglycans during cartilage culture in 'sulfate free' medium.

Incorporation of radiolabeled sulfate into glycosaminoglycans is a widely accepted assay to measure the rate of proteoglycan synthesis. Although glycosaminoglycan synthesis is dependent on the quantity of inorganic sulfate available to proteoglycan synthesizing cells, 'sulfate free' medium is regularly used in studies regarding proteoglycan synthesis. In this study murine patellar cartilage glycosaminoglycans synthesized under 'sulfate free' conditions were compared with those synthesized at physiological sulfate concentration. Under 'sulfate free' conditions synthesis was not only decreased but low sulfated glycosaminoglycans were made that were not synthesized during incubation at physiological sulfate concentration. The use of 'sulfate free' medium should be avoided in proteoglycan synthesis studies.

Effect of nonsteroidal antiinflammatory drugs on cartilage destruction in antigen induced arthritis in mice.

The nonsteroidal antiinflammatory drugs, salicylate, piroxicam and tiaprofenic acid, and the steroid prednisolone were investigated in a long-term study for their potential detrimental or beneficial effects on joint cartilage in mice with antigen induced monoarthritis. Daily drug treatment over a period of 4-7.5 weeks did not affect the histological characteristics of normal joints at all. Articular chondrocyte synthetic activity was even stimulated after salicylate and tiaprofenic acid treatment, but the significance of this finding is not yet clear. Cartilage damage, caused by inflammation in the knee joint, was neither markedly deteriorated nor attenuated by these drugs. Minor antiinflammatory properties as measured by decrease in edema using 99mTc-uptake and in the change of inflammatory cells were only evident with prednisolone, piroxicam and salicylate.

Effects of NSAIDs on the metabolism of sulphated glycosaminoglycans in healthy and (post) arthritic murine articular cartilage.

Several non-steroidal anti-inflammatory drugs (NSAIDs) were studied for their effects on normal and damaged murine articular cartilage, both in vitro and in vivo. In vitro, in the absence of serum, sodium salicylate caused significant suppression of 35S-glycosaminoglycan (GAG) synthesis, whereas tiaprofenic acid, piroxicam, prednisolone sodium phosphate and several other NSAIDs were without effect. Trypsin-mediated proteoglycan depletion did not change the susceptibility of the articular chondrocyte to these drugs. Similarly, no enhancement of drug effect was seen when arthritic cartilage was taken from an acutely inflamed joint, and prenisolone sodium phosphate even seemed to diminish inflammation-mediated suppression of 35S-GAG synthesis. The short term in vivo effects of some of the drugs were tested in mice with unilateral zymosan-induced arthritis. At day 1 after arthritis induction, in vivo 35S-GAG synthesis by the cartilage of the arthritic joint was decreased to 63%. Only sodium salicylate suppressed in vivo 35S-GAG synthesis in the healthy and arthritic joint to the same extent in both. At day 28, GAG synthesis in the postarthritic joint was enhanced to 160%. This type of cartilage appeared to be more susceptible to drug effects, since all drugs tested showed clear suppression of the augmented GAG production in vivo. Finally, in vivo drug effects were tested on normal and enhanced 35S-GAG degradation, the latter in the zymosan-induced arthritic joint. Both tiaprofenic acid and prednisolone sodium phosphate appeared to suppress degradation in healthy and, to some extent, in arthritic cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of small quantities of sulfate (0-12 nmol) in serum, urine, and cartilage of the mouse.

The colorimetric benzidine method of K. S. Dodgson and B. Spencer (1953, Biochem. J. 55, 436-440) for the measurement of inorganic sulfate can be scaled down about 100 times by using disposable 96-well microplates instead of individual cuvettes. Ten-microliter samples of serum and urine, derived from mice, can be analyzed in a simple, rapid, and reliable way without sacrificing the animals. Without prior isolation of sulfated glycosaminoglycans, ester sulfate in mouse patellar cartilage is liberated quantitatively as inorganic sulfate upon acid hydrolysis in 3 M HCl for 16 h at 80 degrees C. To this end the articular cartilage layer of the patella must be separated in toto from the underlying bone. Subsequent hydrolysis in polypropylene tubes gives accurate results. In contrast, hydrolysis in borosilicate glass vials is useless, since nanomoles of sulfate added cannot be recovered adequately. The thin patellar cartilage layer obtained from 10-week-old male mice contains about 5 nmol of sulfate, an amount easily measured with the developed microplate benzidine method.

The effect of salicylate on anatomically intact articular cartilage is influenced by sulfate and serum in the culture medium.

The established, suppressive effect of salicylate on sulfated glycosaminoglycan (sGAG) synthesis by normal articular cartilage was reinvestigated using anatomically intact articular cartilage of the whole mouse patella. Employing the physiological murine sulfate concentration (1.0 mM) sodium salicylate (1-5 X 10(-3) M) caused a dose dependent inhibition of 35S-sGAG synthesis (10-35%). At a lower sulfate concentration (0.4 mM) this inhibition was increased (15-45%) and the suppression was even more pronounced in sulfate deprived medium. This observation stresses the need of using physiological sulfate concentrations in cartilage culture studies. In the presence of 100% serum the therapeutic drug concentration (1-2 X 10(-3) M) had no longer any suppressive effect, either at 1.0 mM or at any lower sulfate concentration. Our data suggest that salicylate has no direct effect on sGAG metabolism in normal articular cartilage in vivo and that adverse effects may be due to the observed salicylate induced lowering of the endogenous sulfate level.

Quantitation of glycosaminoglycan metabolism in anatomically intact articular cartilage of the mouse patella: in vitro and in vivo studies with 35S-sulfate, 3H-glucosamine, and 3H-acetate.

We investigated the usefulness of the whole mouse patella to quantitate the synthesis of the glycosaminoglycan (GAG) backbone and its sulfation by intact murine articular cartilage, both in vitro and in vivo. Using 35S-sulfate, 3H-glucosamine, or 3H-acetate as precursors of GAG synthesis, it was found that more than 90% of the incorporated radioactivity was confined to the patellar cartilage layer compared to the whole patella. Overnight papain digestion was enough to liberate more than 95% of the incorporated radiolabels, except for 3H-acetate for which 15-25% was not digestible. Comparison of radioactivity in the patella and that in quantitatively isolated GAGs revealed that for 35S-sulfate incorporation studies the whole patella can be used as a reliable measure for sulfated GAG synthesis. The situation was different for the GAG backbone precursors 3H-glucosamine and 3H-acetate; more than 50% of the 3H labels were incorporated into compounds other than GAGs or non-covalently associated with matrix components. Hence, in studying GAG-backbone metabolism, polysaccharides must be isolated quantitatively from cartilage. In vivo studies made it clear that both 35S-sulfate and 3H-glucosamine are incorporated into patellar GAGs in amounts high enough to enable proper quantitation and that the route of administration (intraperitoneally or intravenously) is of minor importance. Due to its low specificity for cartilage GAGs, 3H-acetate is not suitable for such studies.

Salicylate-induced depletion of endogenous inorganic sulfate. Potential role in the suppression of sulfated glycosaminoglycan synthesis in murine articular cartilage.

Sodium salicylate has been shown to suppress glycosaminoglycan (GAG) synthesis by articular hyaline cartilage in vitro. We investigated the in vivo effect of sodium salicylate on murine patellar cartilage, using incorporation of intraperitoneally administered 35S-sulfate as a measure of sulfated GAG synthesis. Our results indicated that a single dose of sodium salicylate (200 mg/kg) inhibited in vivo sulfated GAG synthesis by 56%, compared with controls, and had no effect on sulfated GAG breakdown. A striking finding was that sodium sulfate treatment reduced the serum concentration of inorganic sulfate from 1.1 mM to approximately 0.3 mM, and that this serum reduction was associated with a twofold increase in urinary excretion of sulfate. Using anatomically intact patellar cartilage, in vitro studies clearly showed that, in concentrations reached in vivo (greater than or equal to 1 mM), salicylate suppressed murine chondrocyte GAG synthesis. However, in the presence of serum, the effects of 1 mM salicylate were abolished. We also found that sulfated GAG synthesis was clearly inhibited at low concentrations of sulfate (less than 0.5 mM). Our data indicate that sodium salicylate can suppress articular chondrocyte sulfated GAG synthesis in vivo, and that this effect may particularly be due to a drug-induced reduction of endogenous sulfate.