The effect of ascorbic acid oxidation on the incorporation of sulfate by slices of calf costal cartilage

A marked inhibition of the incorporation of S³⁵-sulfate by normal calf costal cartilage was produced by potassium ascorbate in the presence of catalytic amounts of cupric ions. The effect of the various components of the ascorbic acid oxidizing system (potassium ascorbate, cupric ions, cuprous ions, hydrogen peroxide, dehydroascorbic acid) was investigated. The results of experiments in which hydrogen peroxide, catalase, or sodium azide were used singly or in combination suggest that the inhibition produced by the ascorbic acid oxidizing system is due, to a considerable extent, to the production of hydrogen peroxide. Dehydroascorbic acid was also found to inhibit the incorporation of S³⁵-sulfate by cartilage slices. However, the gradual fall in pH which resulted from the addition of dehydroascorbic acid could account to a large extent for the inhibitory effect observed because the incorporation of S³⁵-sulfate by cartilage slices decreases sharply as the pH is lowered. The incorporation of S³⁵-sulfate by cartilage slices is inhibited also by increasing the concentration of phosphate.     

Comparison of sulfate metabolism in costal cartilage of normal and 'little' mice

C57BL/6J and mutant 'little' (lit/lit) mice c. 50 days of age were injected with doses of [35S]sulfate proportional to their body weight. Despite the diminished growth rate of lit/lit mice compared with normal mice at this age, uptake of radioactivity per unit mass of cartilage was similar for both mouse types, confirming previous data. Additional experiments with these mice established that the similarity of sulfate uptake could not be accounted for by differences in the location of bound sulfate or (for females) by differences in cartilage cellularity. Investigation of sulfate loss by costal cartilage in vivo indicated that cartilage degradation occurred at a greater rate in lit/lit mice than in normally growing mice. These latter data suggest that growth hormone, which is lacking in lit/lit mice, may in part regulate skeletal growth (at least for female mice) by inhibiting degradation of cartilage.     

Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.

Since the original work by Sweeley et al. (1) the silylation and subsequent gas-liquid chromatography of simple sugars has been well documented (2–5). The silylation of their biochemically important derivatives, notably sugar phosphates, has proven more difficult. Wells et al. (6) premethylated the phosphate with diazomethane in methanol before silylating the sugar group with the usual reagents [pyridine, hexamethyldisilazane (HMDS), and trimethylchlorosilane (TMCS)], Hashizume and Sasaki (7) refluxed the sugar phosphates in the reaction mixture for 1 hr to effect complete silylation, while Eisenberg and Bolden (8) achieved similar results by heating in a sealed vial at 100°C for 10 min. The latter researchers noted that glucose 1-phosphate tended to decompose under their conditions to give one major and two minor peaks. Horning et al. (9) and, later, Sherman et al. (10) used bis(trimethylsilyl)acetamide (BSA) to silylate sugar phosphates, but Pierce (11) has noted that BSA tends to give inconsistent silylation of simple sugars and we have also noted this in our work.In 1969, Ellis (12) reported on the use of a silylation system using dimethylsuifoxide (DMSO) or dimethylformamide (DMF) as the reaction solvent rather than pyridine. The silylated derivatives are not soluble in either of these solvents and they form a second, upper liquid layer (in 10 min in the case of DMSO, 18 hr for DMF). The silylated sample is thus concentrated and this avoids the necessity of removing the solvent [with its attendant problems (10)] after silylation.This paper reports a modification and extension of the silylation system described by Ellis which allows rapid, quantitative silylation of sugars and sugar phosphates at room temperature, i.e., the addition of cyclohexane to the silylation mixture to promote the silylation of sugar phosphates and other biologically important acids. Also reported is the first use of SE-52 stationary liquid phase for the glc of sugar phosphates.     

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Clarified cell-free extracts were prepared from rapidly dividing $\text{Bacillus subtilis}$ cells and from rabbit liver cells. These extracts were treated with [³H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the $\text{B. subtilis}$ extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.     

Suppression of active cross-bridge motions of isolated thick myofilaments in suspension by phenylmethylsulfonyl fluoride

Studies of photoelectron count autocorrelation function of light scattered from suspensions of thick filaments of Limulus telson muscle and scallop striated adductor muscle reveal that Ca2+ can activate cross-bridge motions of these isolated filaments. By treating suspensions of activated filaments with phenylmethylsulfonyl fluoride (PMSF), we can suppress active cross-bridge motions but not affect the Ca2+-dependent ATPase activity.     

Dielectric characterization of costal cartilage chondrocytes

Background

Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

Methods

Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes.

Results

The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance.

Conclusions

We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues.

General significance

The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.     

The effect of calcification on the structural mechanics of the costal cartilage

The costal cartilage often undergoes progressive calcification with age. This study sought to investigate the effects of calcification on the structural mechanics of whole costal cartilage segments. Models were developed for five costal cartilage specimens, including representations of the cartilage, the perichondrium, calcification, and segments of the rib and sternum. The material properties of the cartilage were determined through indentation testing; the properties of the perichondrium were determined through optimisation against structural experiments. The calcified regions were then expanded or shrunk to develop five different sensitivity analysis models for each. Increasing the relative volume of calcification from 0% to 24% of the cartilage volume increased the stiffness of the costal cartilage segments by a factor of 2.3–3.8. These results suggest that calcification may have a substantial effect on the stiffness of the costal cartilage which should be considered when modelling the chest, especially if age is a factor.     

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.     

Protection by acyl-carnitines and phenylmethylsulfonyl fluoride of rat heart subjected to ischemia and reperfusion

Perfusion of rat hearts according to the Langendorff technique with micromolar concentrations of palmitoylcarnitine or millimolar concentrations of phenylmethylsulfonyl fluoride protect the heart from deterioration by reperfusion after total-ischemia. This is based on the retention of the cytosolic enzymes determined (lactate dehydrogenase, glycogen phosphorylase and glycogen synthase) and of myoglobin, as well as on the resumption of contractile activity. Palmitoylcarnitine, like phenylmethylsulfonyl fluoride, could protect through plasmamembrane stabilization, since more hydrophylic compounds had no effect.     

Influence of phenylmethylsulfonyl fluoride on anandamide brain levels and pharmacological effects

The endogenous cannabinoid anandamide produces cannabimimetic effects similar to those produced by delta9-tetrahydrocannabinol (delta9-THC), but has a much shorter duration of action due to its rapid metabolism to arachidonic acid and polar metabolites via action of fatty acid amide hydrolase (FAAH). Our earlier observations that anandamide's effects persisted after brain levels of anandamide itself had substantially dropped prompted us to examine the influence of the irreversible amidase inhibitor, phenylmethyl sulfonyl fluoride (PMSF), on the brain levels and pharmacological effects of anandamide. As shown previously, pretreatment with PMSF resulted in a leftward shift of the anandamide dose effect curves for antinociception and hypothermia in male mice. Brain and plasma levels of anandamide, arachidonic acid and polar metabolites peaked at 1 min after i.v. injection with 3H-anandamide and remained high at 5 min post-injection, with levels falling sharply thereafter. Pretreatment with PMSF (30 mg/kg, i.p.) prior to an injection of 1 or 10 mg/kg 3H-anandamide resulted 5 min later in enhanced brain levels of anandamide compared to those obtained with 3H-anandamide plus vehicle injection. Levels of arachidonic acid and polar metabolites in brain were not significantly increased. The clear correspondence between brain levels of anandamide following pretreatment with PMSF and pharmacological activity suggests that this parent compound is responsible for the antinociception and hypothermia that occurred 5 min after injection. These results further suggest that metabolite contribution to anandamide's effects, if any, would occur primarily at later times.     

Radiomodfication of xanthine oxidoreductase system in the liver of mice by phenylmethylsulfonyl fluoride and dithiothreitol

The widely distributed xanthine oxidoreductase (XOR) system has been shown to be modulated upon exposure of animals to ionizing radiation through the conversion of xanthine dehydrogenase (XDH) into xanthine oxidase (XO). In the present work, radiomodification of the XOR system by phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) was examined using female Swiss albino mice which were irradiated with gamma rays at a dose rate 0.023 Gy s(-1). PMSF, a serine protease inhibitor, and DTT, the sulfhydryl reagent, were administered intraperitoneally prior to irradiation. The specific activities of XDH and XO as well as the XDH/XO ratio and the total activity (XDH+XO) were determined in the liver of the mice. The inhibition of XO activity, restoration of XDH activity, and increase in the XDH/XO ratio upon administration of PMSF were suggestive of irreversible conversion of XDH into XO mediated through serine proteases. The biochemical events required for the conversion were probably initiated during the early phase of irradiation, as the treatment with PMSF immediately after irradiation did not have a modulatory effect. Interestingly, DTT was not effective in modulating radiation-induced changes in the XOR system or oxidative damage in the liver of mice. The DTT treatment resulted in inhibition of the release of lactate dehydrogenase. However, the protection appears to be unrelated to the formation of TBARS. On the other hand, the presence of PMSF during irradiation inhibited radiation-induced oxidative damage and radiation-induced increases in the specific activity of lactate dehydrogenase. These findings suggest that a major effect of ionizing radiation is irreversible conversion of xanthine to xanthine oxidase.     

Hormonal control of sulfate uptake by branchial cartilage of coho salmon: role of IGF-I.

The direct hormonal control of sulfate uptake by cartilage matrix of coho salmon was examined by exposing branchial cartilage to 1 microCi.ml-1 35SO4 for 48 hours at 15 degrees C in a defined medium. Sulfate uptake occurred primarily in cartilage (rather than bone) and the amount of specific uptake was similar in epibranchial and ceratobranchial cartilages. Intact and hypophysectomized coho salmon starved for 22 days had equivalent in vitro sulfate uptake, which in both cases were 30% of the uptake seen in branchial cartilage of fed, intact controls. In branchial cartilage from starved coho salmon, in vitro exposure to recombinant bovine insulin-like growth factor I (rbIGF-I) at 1, 10, 100, and 1,000 ng.ml-1 caused a dose-dependent increase in sulfate uptake, with a maximum 3-fold increase over control at 1,000 ng.ml-1 rbIGF-I. Coho salmon insulin (1, 10, 100, and 1,000 ng.ml-1) resulted in a maximum 30% increase in sulfate uptake at the highest dose. Growth hormone and triiodo-L-thyronine had no direct effect on in vitro sulfate uptake. The results indicate that IGF-I has direct effects on coho salmon cartilage and may be an important regulator of growth in salmon and other teleosts.     

An investigation of ageing in human costal cartilage

Summary Changes in human costal cartilage with increasing age (2–81 years) have been studied in the optical and electron microscope using routine and histochemical techniques.Concurrent with increasing age, chondrocytes undergo degeneration which is characterized initially by the accumulation of lipidic material within cells and, subsequently, by the formation of a halo around degenerating chondrocytes. The halo material is composed of electron dense bodies, amorphous material, and collagen fibrils. Both electron dense bodies and the amorphous material are of cellular origin and they have similar histochemical responses.Using histochemical techniques in the optical and in the electron microscope, it has been shown that chondroitin sulfate decreases with increasing age, while a hyaluronidase resistant material (presumably keratan sulfate) increases, initially in the central zone, and subsequently in the peripheral zones. Hyaluronidase resistant material is minute or absent in the central zone of aged cartilage.The genesis of collagen fibrils progresses from thin unbanded collagen-like fibrils in the pericellular lacunae of chondrocytes in young specimens to thick fibrils (sometimes in excess of 0.5 ) with a period of 640 Å in ageing cartilage. Aggregation of collagen fibrils seems to be related at least initially to the preponderance of matrix granules and beaded filaments which have been shown to originate intracellularly in vacuoles formed in degenerating mitochondria. Both of these structures contain glycosaminoglycans and, with increasing age, glycosaminoglycans decrease while collagen fibrils aggregate. In old age, the amorphous material, and possibly the content of disrupting electron dense bodies, seem to give origin to some collagen fibrils. This and other mechanisms of formation of collagen fibrils have been observed and they are discussed.Calcification of the matrix increases with increasing age and this agrees with previous findings.Supported by grants from the Italian National Research Council. — The authors are indebted to Miss Giuliana Silvestrini and to Mr. Lucio Virgilii for their expert and extensive technical assistance. — To Dr. A. Ascenzi, Director 1° Istituto di Anatomia e Istologia Patologica, and to Dr. C. Cavallero, Director, 2° Istituto di Anatomia e Istologia Patologica, Università di Roma, the senior author would like to express his appreciation for the use of equipment and facilities pursuant to this investigation, while on sabbatical leave from the University of California, Irvine, College of Medicine. — We wish to extend our thanks to the Italian National Research Council for supporting this study.On sabbatical leave from the University of California, Irvine, College of Medicine.     

Turnover of proteoglycans in guinea pig costal cartilage

The turnover in vivo of proteoglycans of guinea pig costal cartilage was investigated using Na₂³⁵SO₄ as precursor. Proteoglycans were extracted with guanidine · HCl, at both low and high ionic strength, and purified and fractionated by ultracentrifugation in CsCl gradients under associative and dissociative conditions. The results suggest that the sulfate is incorporated into macromolecules of at least two major metabolic pools with half-lives of about 3 days and about 60–70 days, respectively. Molecules with a fast turnover were enriched in the low ionic strength extracts and in fractions containing small, nonaggregated proteoglycans. No substantial evidence was found for a precursor-product relationship between different fractions.     

Kinetics of sulfate uptake by yeast

Uptake of sulfate by yeast requires the presence of a metabolic substrate and is dependent on the time during which the cells have been metabolizing in the absence of sulfate. At low concentrations of sulfate, uptake can be described by simple saturation kinetics. Uptake of sulfate is accompanied by a net proton influx of 3 H⁺ and an efflux of 1 K⁺ for each sulfate ion taken up. Divalent cations stimulate sulfate uptake at low concentrations of sulfate; the maximal rate of uptake is not significantly affected but $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ is lowered. Stimulation by divalent cations shows an optimum at a cation concentration of about 4 mM. Monovalent cations are less effective, trivalent cations are more effective in stimulating sulfate uptake. The results are qualitatively in accordance with the notion, that the effect of cations is due to an effect via the surface potential.     

The effect of aging on the synthesis of hexosamine-containing substances from rat costal cartilage. A decrease in sulfation of chondroitin sulfate with aging.

The amount of glycosaminoglycan (GAG) in dry costal cartilage tissue of rats decreased with aging, while the GAG content in mg DNA (unit cartilage cell) remained the same with aging. These results can be explained by the finding that the total number of cartilage cells decreased with aging. Electrophoretic analysis showed that chondroitin 4-sulfate was the major GAG in rat costal cartilage of various ages. Rat costal cartilage of different ages was incubated with radioactive precursors, and newly synthesized GAG was prepared and the radioactivity analyzed to determine the biosynthetic activity. As to changes in the radioactivity uptake with aging per mg dry cartilage tissue, aging influenced [35S]sulfate incorporation into GAG more significantly than [3H]glucosamine incorporation into GAG. There was a significant decrease in the specific radioactivity of [35S]sulfate per mg DNA (unit cartilage cell), whereas the specific radioactivity of [3H]glucosamine per mg DNA did not change significantly with aging. Both the total sulfotransferase activity and the specific activity per mg DNA decreased significantly with aging. Analysis of disaccharide units formed after chondroitinase ABC digestion of labeled GAG isolated from young and old cartilage showed that the percentage of incorporation of [3H]glucosamine into deltaDi-OS increased significantly with aging. These results suggested that the appearance of nonsulfated positions in the structure of the chondroitin sulfate chain increased with aging. On the basis of gel chromatography on Bio-Gel A-1.5 m no significant difference in the approximate molecular size of chondroitin sulfate was observed between the young and old GAG samples. The present study indicated that the sulfation of chondroitin sulfate chains from rat costal cartilage decreased with the process of aging.     

Proteinase participation in chromosome radiation damage. Research on the action of phenylmethylsulfonyl fluoride

Phenylmethylsulfonyl fluoride, a chromatin proteinase inhibitor, caused a nearly twofold diminution of the cytogenetic injury and a twofold increase in the rate of DNA repair in gamma-irradiated (3-15 Gy) Chinese hamster fibroblasts. The effect of the inhibitor was mainly exhibited by a rapidly repaired (for 15-20 min) component of the cytogenetic damage. A simultaneous treatment with phenylmethylsulfonyl fluoride and nicotinamide did not influence the effect of the proteinase inhibitor under study. The results obtained are indicative of poly (ADP-ribosylation)-independent contribution of chromatin proteinases to radiation-induced chromosome mutagenesis.