Transfection of rabbit articular chondrocytes by the calcium phosphate procedure: Improvement of efficiency and reproducibility

The understanding of cartilage disorders relies on the possibility of studying mechanisms which monitor the regulation of matrix protein genes through introducing efficiently and in a reproducible manner these genes, or their regulatory regions, into cells. To this end, we attempted to improve the transfection efficiency of rabbit articular chondrocytes by the calcium phosphate procedure. Transfection efficiencies were assessed by measuring the expression of the Lac Z reporter gene encoding -galactosidase using anin situ staining (X-gal staining) and an enzymatic assay (-galactosidase assay).Results revealed that addition of 4 U ml^–1 of hyaluronidase before and during transfection increases by 2 to 4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, we demonstrated that the use of a giant calcium phosphate DNA coprecipitate gives a higher transfection efficiency and much more reproducible results than those obtained with classical small volumes of precipitates.Abbreviations CPRG Chlorophenol red--D-galactopyranoside - EDTA Ethylenediaminetetraacetic acid - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - HBS Hepes buffered saline - RAC Rabbit articular chondrocytes - X-gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside     

Estradiol protects cultured articular chondrocytes from oxygen-radical-induced damage

Osteoarthritis (OA) is aggravated in menopausal women possibly because of changed serum estrogen levels. Estradiol has been postulated to affect oxidative stress induced by reactive oxygen species (ROS) in articular chondrocytes. We generated ROS in cultured bovine articular chondrocytes by incubating them with combined Fe₂SO₄, vitamin C, and hydrogen peroxide. The release of thiobarbituric-acid-reactive substances (TBARS, lipid peroxidation) and lactate dehydrogenase (LDH, membrane damage) was measured photometrically. Various estradiol doses and vitamin E, serving as control with an established anti-oxidative capacity, were applied either upon each exchange of medium and during radical production (strategy 1) or only during radical production (strategy 2). In chondrocytes incubated according to strategy 1, the production of TBARS and LDH release were significantly suppressed by 10^–10–10^–4 M estradiol or by vitamin E. Under strategy 2, the production of TBARS was significantly suppressed at estradiol concentrations higher than 10^–6 M, whereas LDH release was inhibited at concentrations of 10^–6–10^–4 M. Vitamin E showed no significant effects. As repeated application of estradiol and vitamin E produced the best results, estradiol, like vitamin E, was speculated to accumulate in the plasma membrane and to decrease membrane fluidity resulting in protection against lipid peroxidation (non-genomic effect). Thus, in contrast to the neuroprotective effect of 17-estradiol in supraphysiological doses reported recently, the anti-oxidative potential of estradiol appears to protect articular chondrocytes from ROS-induced damage when the hormone is given repeatedly in a physiological range. Decreased estradiol levels may therefore contribute to menopausal OA in the long term.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats

Summary Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.     

Effects of feeding and starvation on growth and swimming activity in an obligatory air-breathing fish

Reared in (tubular) aquaria containing different depths of water, Ophiocephalus striatus (0.7 g, 4.5 cm body length), an obligatory air-breathing tropical fish, swam long or short distances to enable themselves to exchange atmospheric air. In each tested depth (2.5, 5.0, 15.5, 31.0 and 40.0 cm) series, one group was starved, while the other was fed ad libitum twice a day on fish muscle. In the shallowest water (2.5 cm depth), the feeding group surfaced 1,294 times, travelling 64.7 m at an energy cost of 20.4 mg dry fish substance/g live fish/day, against those exposed to the deepest water (40 cm depth), which expended 35.8 mg/g/day, swimming 1,503.4 m on 1,879 visits to the surface. The starving group surfaced only 482 times, travelling 24.1 m at an expense of 5.8 mg/g/day in the shallowest water, while those at 40 cm depth surfaced 504 times, swimming 403.2 m at an energy cost of 7.4 mg/g/day. Owing to the sustained swimming activity and the consequent fatigue, the test individuals belonging to both groups in all the tested series hang to the surface for a definite interval, repaying the O₂ debt. Observations were also made to assess the duration of hanging to precisely estimate the distance travelled. Irrespective of changes in depths of water, the duration of hanging to surface was only 3.0 hr/day for the feeding groups, while it was as much as 15.5 hr/day for the starving groups. The maximum sustained metabolic level of O.striatus reared in 40 cm depth was equivalent to 1.23 ml O₂/g/hr, which is about 2 times higher than the value reported for the active metabolism of swimming Oncorhynchus nerka at 15°C in Brett's (1964) respirometer. O.striatus reared in 2.5 cm depth fed 32.0 mg and converted 6.7 mg dry food/g live fish/day, while those exposed to the deepest water fed 49.1 mg, but converted only 5.5 mg/g/day. Culturing obligatory air-breathing fishes in shallow waters will be advantageous.     

Enzyme activity measurements on isolated organs of Brachionus plicatilis (Rotifera)

A method is described by which the integument of Brachionus plicatilis, together with its intracellular lamina, is quickly dissolved before other parts or tissues of the animal are destroyed. After removing the integument several parts of the body can be separated and fractionated in a more or less intact state by centrifugation in a Percoll gradient. The measurement of enzyme activities has indicated that this procedure might provide a way of localizing enzymes within the rotifer body.     

Effects of Lovastatin and Pravastatin on Amyloid Processing and Inflammatory Response in TgCRND8 Brain

Previous studies suggest that treatment with statins reduce beta amyloid (Abeta) deposition in brains of mouse models of Alzheimer's disease (AD) and may reduce the prevalence of AD in humans. Since lipophilicity influences the biological efficacy of statins, we compared the effects of lovastatin, a lipophilic statin, to effects of the hydrophilic pravastatin on amyloid processing and inflammatory responses in brain. Three-month old TgCRND8 mice expressing mutant human amyloid precursor protein (mHuAPP) were treated daily with various doses of either statin. After 1 month, levels of cerebral soluble and fibrillar Abeta peptides, soluble sAPPalpha, and inflammatory cytokines were measured. Both statins caused dose-dependent reductions in total Abeta peptides with parallel increases in total sAPPalpha. At all doses, slightly greater effects were observed with lovastatin than with pravastatin. In contrast, only lovastatin significantly increased levels of IL-1beta and of TNFalpha in a dose-dependent manner. Lovastatin, but not pravastatin, decreased succinic dehydrogenase and increased lactate dehydrogenase activities in skeletal muscle and increased TUNEL staining in liver. Our data demonstrate that both statins shift the balance of APP processing from excessive beta-toward the normal alpha-cleavage while reducing the total amyloid burden in TgCRND8 brain and that lovastatin, but not pravastatin, potentiates cerebral inflammation and is associated with liver and muscle histotoxicity in these animals. These data show that pravastatin can reduce amyloid burden without potentiating inflammatory responses in brain and, therefore, may have a wider dose-range of safety than have lipophilic statins in the treatment or prevention of AD.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Development and isoproterenol-induced regulation of adrenoceptor binding in cultured rat neocortical explants is seen only with the beta-1, not with the beta-2 subtype

The presence and time-course of -adrenoceptor density in cultured explants of neocortex obtained from 6-day-old rat pups were investigated using a [¹²⁵I]ICYP binding assay. A delayed, but more pronounced, increase in the receptor expression was observed as compared to the situation previously described in vivo. These changes only occurred for the ₁-subtype of the receptor, whereas the ₂-subtype binding remained constant up to 3 weeks in vitro. The delay of ₁-adrenoceptor expression may be due to the incomplete presence of the proper maturational input, and the late enhancement of receptor expression to upregulation related to the absence in vitro of noradrenergic input. Decreased -adrenoceptor levels could be induced by chronic treatment of the -agonist isoproterenol (1 M) introduced either for 3 or 13 days. Again, changes in density were found only for the ₁-adrenoceptor binding sites. There is no reduction of receptor density following return to control conditions for 10 days after a 3-day treatment with isoproterenol, demonstrating the ability of this model to attain its final receptor density notwithstanding the developmental insult.Special issue dedicated to Dr. Robert Balázs     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Fatty acid composition of vitellogenin from four teleost species

The fatty acid compositions of vitellogenin and liver from cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and wolffish (Anarhichas lupus) were determined. Vitellogenin was isolated from plasma of estradiol-17-treated fish by precipitation with EDTA-Mg²⁺ and distilled water or by high-performance ion-exchange chromatography. In all investigated species, vitellogenin contained 16–18% (w/w) lipid, in which polyunsaturated fatty acids, predominantly 20:5 (n-3) and 22:6 (n-3), comprised about 50% of the total fatty acids. The proportions of saturated, monounsaturated, polyunsaturated and (n-3) fatty acids in vitellogenin of the different species were generally similar, although the relative content of specific fatty acids was distinctive for each species. The distribution of fatty acids in total lipids of vitellogenin was highly consistent among individual females of each species. In contrast, liver fatty acid composition varied considerably, both within and between species. Altogether, the differences in the fatty acid composition of vitellogenin and liver from each species indicate that a specific selection of fatty acids occurs during the lipidation of vitellogenin.Abbreviations BHT butylated hydroxytoluene - E-17 estradiol-17 - EDTA ethylenedinitrilo tetra-acetic acid disodium salt dihydrate - FA fatty acids - FAME fatty acid methyl esters - HDL high density lipoproteins - PUFA polyunsaturated fatty acids - SD standard deviation - TLC thin-layer chromatography - VHDL very high density lipoproteins - VLDL very low density lipoproteins - v/v volume per volume - w/v weight per volume - w/w weight per weight     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Influence of growth conditions on the production of xylanolytic enzymes by Trametes trogii

Various nitrogen and carbon sources were examined as inducers of the production of endoxylanase and -xylosidase by Trametes trogii. T. trogii grown on xylan plus crystalline cellulose provided supernatants with the highest enzymatic activities. Organic nitrogen sources (especially asparagine and casamino acids) were the best for enzyme production. The increase in xylan concentration stimulated endoxylanase production whereas significant differences were not attained in -xylosidase production with more than 5g xylan/l in the culture medium. pH 4.0 was optimal for endoxylanase production, while -xylosidase production was maximum at pH 5.5. Temperatures in the range of 23–28°C stimulated enzyme production. The endoxylanase activity in the crude culture filtrate was greatest at 50°C and pH around 5.0. The optimum pH and temperature for -xylosidase activity were 5.5 and 50°C respectively.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.