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Biomedical subjects

J de Lange

Publications and source records attributed to J de Lange.

35 records · Page 2Linked to original sources
PubMed1994

Analysis of changes in proteoglycan content in murine articular cartilage using image analysis.

The extracellular matrix of articular cartilage consists mainly of type II collagen and large aggregating proteoglycan (aggrecan). During arthritis and other joint diseases, the proteoglycan (PG) level of cartilage matrix is diminished, leading to impairment of normal joint function. A new method is described for measuring the changes in PG content of murine articular cartilage. The method is based on the automated densitometric analysis of patellar cartilage of standard, safranin O-stained sections of whole murine knee joints. It appeared to be possible to measure optical density in parallel layers of articular cartilage with high reproducibility. Approximately 25 sections can be evaluated within 1 h. Measuring a single section 10 times resulted in a coefficient of variation (CV) of 0.1-1.4%. A mean CV of 5-14% was calculated when a group of 18 sections was analyzed in quintuplicate. To validate the procedure, changes in PG content induced by arthritis or by intra-articular injection of TGFbeta-1 were analyzed by the image analysis method, the dimethylmethylene blue (DMB) assay and by visual grading. Although not a quantitive method, the newly developed image analysis method appeared to be more sensitive in detecting significant change in PG content of murine articular cartilage than the DMB method or visual grading. The image analysis method makes it possible to measure changes in PG content of specific areas of articular cartilage with higher sensitivity than the DMB method and eliminating the bias inherent to visual grading by human observers.

Animals↗
PubMed1992

Therapeutic efficacy of HI-6 in soman-poisoned marmoset monkeys.

The therapeutic efficacy of the oxime HI-6 against intoxication with the irreversible cholinesterase (ChE) inhibitor soman was tested in marmoset monkeys. Five out of six marmosets, intoxicated with 5 x LD50 soman and treated immediately with diazepam (0.2 mg.kg-1 iv) and 15 sec later with atropine (0.5 mg.kg-1 im) and HI-6 (50 mg.kg-1 im), survived for more than 24 hr. One of these animals died after 4 days. In the HI-6-treated marmosets blood ChE activity was inhibited at a rate slower than that in three animals treated similarly but with saline instead of HI-6. The latter marmosets died within 8 min after soman. HI-6 achieved its plasma peak 5 min after injection and was eliminated with a t1/2 of about 40 min. In a second experiment similarly treated marmosets were euthanized at 5 min (three saline-treated animals) or at 10 min (three HI-6-treated animals) after the soman intoxication to enable the determination of acetylcholinesterase (AChE) activities in diaphragm and brain tissue. In addition, in these animals blood AChE and butyrylcholine esterase (BuChE) activities were determined. Low AChE activities were encountered in diaphragms and brains. These levels were not significantly different between saline- and HI-6-treated marmosets. In vitro treatment with HI-6 at 40 min after soman still led to an increase of the AChE activity, which was significant in diaphragm, suggesting that postmortem AChE inhibition had occurred. The ratio of AChE to BuChE in blood was significantly enhanced in HI-6-treated animals, indicating that HI-6 preferentially reactivated AChE. It is concluded that (i) HI-6 is an effective treatment against soman poisoning in marmosets and (ii) AChE reactivation or protection by HI-6 contributed to the survival of the animals.

Acetylcholinesterase↗
PubMed1992

The isolated blood-perfused pig ear: an inexpensive and animal-saving model for skin penetration studies.

To overcome most of the disadvantages of current models to investigate percutaneous penetration of drugs or toxic substances, a model is proposed here based on the isolated pig ear, which is obtained at the slaughterhouse, and perfused with oxygenated blood from the same pig. To determine the viability of the preparations, we measured glucose consumption and lactate production as metabolic parameters, Na+ and K+ ions, as well as lactate dehydrogenase activity in blood as markers for cell damage, whereas vasomotor reactivity was assessed by administering noradrenaline and isoxsuprine. After 60 min of equilibration, only insignificant changes in these parameters were observed during the subsequent 3-hr test period (longer periods were not tested). A slight weight increase was noted during the total period 4 hr, presumably due to slight edema formation. On the basis of several types of measurements, such as in vivo blood flow and ear temperature and in vitro glucose metabolism, standard procedures were developed. It is concluded that this technique offers an easy to handle, cost-efficient, and animal-saving model for skin penetration studies that lacks most of the disadvantages of existing models.

Animal Testing Alternatives↗
PubMed1991

Therapy of organophosphate poisoning in the rat by direct effects of oximes unrelated to ChE reactivation.

Isolated rat diaphragm preparations treated with soman or with the irreversible and oxime resistant cholinesterase (ChE) inhibitor S27 showed a considerable recovery of neuromuscular transmission (NMT) during incubation with the (bis)pyridinium oximes HI-6, HGG-12, P2S and obidoxime. In the soman-treated preparations this NMT recovery was predominantly caused by reactivation of acetylcholinesterase (AChE) but in the S27-treated preparations it was caused by a direct (pharmacological) effect unrelated to enzyme reactivation. Atropinized rats were artificially ventilated after injection with 3 x LD50 soman for 3 h and then treated with HI-6, i.e. at a time when oxime reactivation of soman inhibited ChE is no longer possible. Nevertheless, these rats started to breathe spontaneously and 50-60% survived more than 24 h, whereas all control animals (saline instead of HI-6) died within 10 min after artificial ventilation was terminated. In such animals no significant reactivation of ChE activity at various time intervals following HI-6 treatment was found, either in the diaphragms or in the brains. There was a significant amount of NMT (50%) in vitro in diaphragms obtained from these animals. This NMT did not improve in vitro in the presence of HI-6 and was not inhibited by soman administered to the medium. It is concluded that in this case the NMT found was based on synaptic adaptation to the continued inhibition of ChE and that the survival of the animals might be due to a combination of this synaptic adaptation and central direct effects of HI-6.

Animals↗
PubMed1989

In vitro degradation of the stereoisomers of soman in guinea-pig, mouse and human skin.

The fate of the four stereoisomers of soman [C(-)P(+), C(+)P(+), C(+)P(-) and C(-)P(-)] was studied by incubating 10 microM C(+/-)P(+/-)-soman at pH 7.4 and 37 degrees for various periods in the presence or absence of homogenates (1:10 and 1:20 w/v) of guinea-pig, mouse or human skin. The remaining concentrations of the soman isomers were determined gas chromatographically. Similar rates of spontaneous (non-enzymatic) hydrolysis (K = 0.005 min-1) were found for the four isomers of soman. Hydrolysis of the toxic (C(+/-)P(-)-isomers is not accelerated in the presence of the skin homogenates. In contrast, the non-toxic C(+/-)P(+)-isomers are enzymatically hydrolysed. As the amount of proteins present in the homogenates varied the rate constants for enzyme hydrolysis per protein concentration were calculated. Except for the high hydrolysis rate constant of greater than 0.127/min.g.l for C(+)P(+) in human skin, these values were almost similar (0.031-0.045/min.g.l) for the skin homogenates tested. Irreversible binding sites for the four soman-stereoisomers are only found in homogenates of mouse skin; 122-195 pmol soman-isomer are bound per mg protein. After preincubation of mouse homogenate with 10 microM soman during 18 hr at 0-4 degrees no further binding of the isomers was detected. It is concluded that skin of the three species tested does not contain enzymes that degrade the toxic C(+/-)P(-)-isomers of soman, whereas phosphorylphosphatase activity for the C(+/-)P(+)-isomers is present in the skin of all three species. Binding sites for all four soman isomers are only present in mouse skin.

Animals↗
PubMed1986

Secretion of lipoprotein lipid and synthesis of fatty acids, cholesterol and triacylglycerol by hepatocytes of fasted-refed rats.

Synthetic rates of fatty acid, cholesterol and triacylglycerols, and contents and secretion of lipoprotein lipids, were determined in hepatocytes of rats fed ad libitum a fat-containing stock diet or of rats fasted for 48 h and then refed for 24 or 48 h with stock diet or with a glucose-rich fat-free diet. When compared with the values for the ad libitum-fed rats, fatty acid synthesis was lower in fasted rats, slightly increased in rats refed with the stock diet, but several-fold elevated after refeeding the glucose-rich fat-free diet. Cholesterol synthesis was decreased in the fasted cells, and restored to the control level upon refeeding either diet. Triacylglycerol synthesis from exogenous oleate was greatly stimulated in the cells of fasted-refed rats above the rate in cells of the ad libitum-fed rats, the increase being considerably higher after refeeding the glucose-rich fat-free diet than the stock diet. The amount of triacylglycerol secreted by the cells was also elevated by the fasting-refeeding treatment, but the difference between the two diets was much less pronounced than seen for the lipids' synthetic rates. This imbalance may underlie the huge accumulation of this lipid observed in the heptatocytes after refeeding the rats for 48 h with the glucose-rich fat-free diet.

Animals↗
PubMed1984

A water-soluble cholesteryl-containing trisgalactoside: synthesis, properties, and use in directing lipid-containing particles to the liver.

The synthesis of a trisgalactoside-terminated cholesterol derivative is described. Tris(galactosyloxymethyl)-aminomethane is coupled to cholesterol by using glycyl and succinyl as intermediate hydrophilic spacer moieties. The resulting cholesteryl ester dissolves easily in water, forming monodisperse micelles. When added to dispersions of liposomes or plasma lipoproteins in water, the substance becomes incorporated rapidly into these structures, causing an increase of their buoyant density. Liposomes or low-density lipoproteins, preloaded with the substance, are rapidly cleared from the circulation and taken up by the liver after intravenous injection in rats. This uptake is inhibited by N-acetylgalactosamine but not by N-acetylglucosamine, indicating the specificity of this process.

Acetylgalactosamine↗
PubMed1983

Concomitant increase in hepatic triacylglycerol biosynthesis and cytosolic fatty-acid-binding-protein content after feeding rats with a cholestyramine-containing diet.

Cholestyramine feeding of rats increased the rate of palmitate and glycerol incorporation into triacylglycerols of isolated hepatocytes. Concomitantly an increase of fatty-acid binding by hepatic cytosolic proteins was observed, which could be attributed to an elevation of the content of the fatty-acid-binding protein (Mr 12000). The involvement of this protein in cholesterol, bile-acid and triacylglycerol metabolism is discussed.

Animals↗
PubMed1983

Bile acids and lipids in isolated rat hepatocytes. II. Source of cholesterol used for bile acid formation, estimated by incorporation of tritium from tritiated water, and by the effect of ML-236B.

After incubation in the presence of tritiated water, incorporation of tritium into cholesterol and into different bile acids was several-fold higher using hepatocytes of cholestyramine-fed rats than that found using hepatocytes of control rats. Labeling of the trihydroxylated cholic and beta-muricholic acids was markedly greater than that of dihydroxycholanoic acid. The total amount of label in all bile acids was 30% or less of that in free cholesterol, in both types of hepatocytes. In combination with the data on bile acids mass production we could calculate the average number (N(a)) of tritium atoms incorporated per molecule of newly-formed bile acid. The experimental values of N(a) for cholic and beta-muricholic acid were compared with values of N(n) or N(o), theoretically predicted if these bile acids were derived entirely from newly made or pre-existent cholesterol, respectively. It was deduced for hepatocytes of cholestyramine-fed rats that the bile acids produced in the first hour of incubation should be totally derived from pre-existent cholesterol, whereas 50% and 25% of the cholic and beta-muricholic acid, respectively, produced during the second and third hours of incubation should be derived from newly synthesized cholesterol. The contribution of newly made cholesterol as substrate for bile acid production was also estimated by using ML-236B. In a concentration of 12 micro M, it depressed cholesterol synthesis by 90% during 1 or 3 hours of incubation of hepatocytes of cholestyramine-fed rats. Mass production of cholic acid was depressed by 25% and that of beta-muricholic acid was not inhibited at all by ML-236B during the first hour of incubation, while they were depressed by 71 and 52%, respectively, during the second plus third hours of incubation. It is concluded that 1) in isolated hepatocytes newly made cholesterol can be a significant substrate for bile acid formation; 2) there are separate cholesterol substrate pools for the productions of cholic or beta-muricholic acid; 3) of the total carbon flux directed into cholesterol synthesis, the major part ends up as free cholesterol and only a minor part as bile acids, even in hepatocytes with a derepressed bile acid production.-Kempen, H. J., M. Vos-van Holstein, and J. de Lange. Bile acids and lipids in isolated rat hepatocytes. II. Source of cholesterol used for bile acid formation, estimated by incorporation of tritium from tritiated water, and by the effect of ML-236B.

Animals↗
PubMed1982

Bile acids and lipids in isolated rat hepatocytes: content, synthesis, and release, as affected by cholestyramine treatment of the donor rats.

Contents of bile acids and lipids, as well as rates of triglyceride synthesis, were determined in isolated hepatocytes from control or cholestyramine-fed rats (denoted below as "control" or "treated" hepatocytes, respectively). During a 3-hr incubation period, total bile acid production was markedly higher in "treated" cells than in "control" cells. With both kinds of cells a marked fall in production rate occurred after the first hour of incubation. Newly produced bile acids appeared in the conjugated form with both kinds of hepatocytes. "Control" cells produced only taurine-conjugated, while "treated" cells made both taurine-conjugated and glycine-conjugated bile acids. However, with exogenous taurine (0.5 mM), the latter cells also produced taurine-conjugated bile acids only. With both kinds of cells, cholic and beta-muricholic acids, but not dihydroxylated bile acids, appeared as newly formed species during the incubation. Addition of dialyzed rat serum to the incubation did not result in a stimulation of bile acid production, with either kind of hepatocytes. "Treated" cells had a slightly higher content of free cholesterol than control cells; contents of other lipids were not different. Fractional release of bile acids and lipids into the medium did not differ between the two kinds of cells. Triglyceride synthesis from added [14C]palmitate (0.5 mM) was 1.8-fold higher in "treated" than in "control" hepatocytes.

Animals↗
PubMed1979

Atlanto axial rotatory fixation--a cause of torticollis.

Atlanto-axial rotatory fixation is a rare but important cause of persistent torticollis. The diagnosis is confirmed by careful radiological examination of the atlanto-axial joint, also employing tomography if necessary. Treatment is skull traction in an attempt to improve the position followed by posterior atlanto-axial fusion to prevent further, and potentially disastrous, displacement. Atlanto-axial dislocation or subluxation should be looked for in all cases of acute torticollis, when reduction by traction will most likely be successful and prevent the development of atlanto-axial rotatory fixation and the associated persistent torticollis.

Atlanto-Occipital Joint↗
PubMed1971

[Glioma cerebri].

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Brain Neoplasms↗