Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with &#103 -cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- &#103 -cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 &#119 g 100 ml &#109 1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- &#103 -cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- &#103 -cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 &#119 g 100 ml &#109 1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- &#103 -cyclodextrin alone.     

Evolutionary Advantages of Participation of Vasopressin instead of Vasotocin in Regulation of Water–Salt Balance in Mammals

In experiments on non-anesthetized Wistar white rats there was studied reaction of kidney to an intramuscular injection of arginine vasotocin or arginine vasopressin at doses from 0.001 to 0.05 µg/100 g body mass on the background of a water load. Water (5 ml/100 g body mass) was administered through a catheter into stomach to suppress secretion of endogenous antidiuretic hormone (ADH). In experiments with water administration, diuresis increased due to a decrease of osmotic permeability of renal tubules and to excretion of osmotically free water, with the constant clearance of sodium ions. Injection of 0.05 µg arginine vasopressin led to a marked decrease of diuresis due to a rise of reabsorption of osmotically free water without elevation of excretion of osmotically active substances. Injection of the same dose of arginine vasotocin resulted in no increase of diuresis; however, reabsorption of osmotically free water and excretion of osmotically active substances including sodium ions were more pronounced. Hence, both vasotocin and vasopressin increased osmotic permeability of the tubular epithelium, but vasotocin, unlike vasopressin, promoted reduction of reabsorption of sodium ions and their loss with urine. A suggestion is made that one of the reasons for replacement in mammals of the molecular ADH forms (vasotocin by vasopressin) was the absence of the pronounced natriuretic effect in arginine vasopressin. This was of crucial significance to preserve sodium ions in the organism, to maintain water–salt balance in animals adapted to the terrestrial life, and to provide not only osmo-, but also volumoregulation.     

Mode of action of sulfoxides in preventing loss of activity of 11β-hydroxylase in cultured bovine adrenocortical cells

Previously, loss of 11β-hydroxylase activity when adrenocortical cells are incubated with the pseudosubstrate cortisol was found to be reduced when the concentration of oxygen was lowered, or when butylated hydroxyanisole (BHA) or dimethyl sulfoxide (Me₂SO) were included in the medium. In the present experiments, we tested the hypothesis that Me₂SO protects 11β-hydroxylase by scavenging OH^? radicals. Substances known to react with OH^? at high rates and non-toxic enough to be used at concentrations of 10–100 mM, including several alcohols, benzoate and radioprotectant thiols, did not prevent loss of activity of 11β-hydroxylase in the presence of 50 μM cortisol. Two of the alcohols, ethanol and glycerol, as well as Me₂SO, were radioprotective in cultured bovine adrenocortical cells. Therefore free OH^? radicals do not appear to be involved in loss of 11β-hydroxylase activity. When sulfoxides other than dimethyl sulfoxide were tested for their ability to protect 11β-hydroxylase in the presence of cortisol, several aryl sulfoxides, particularly dibenzyl sulfoxide, as well as dipropyl sulfoxide, were active at concentrations to $\text{1}\text{200}$ of that required for Me₂SO. Previously, we have demonstrated that 11β-hydroxylase inhibitors, particularly metyrapone, effectively protect against loss of 11β-hydroxylase activity in the presence of pseudosubstrates and therefore we examined whether sulfoxides may act by directly inhibiting 11β-hydroxylase. Me₂SO showed an ED₅₀ for inhibition of 11β-hydroxylase activity of > 1 M, in contrast to its ED₅₀ for protection of 34 mM. For metyrapone, however, the ED₅₀ for inhibition of the enzyme (250 nM) was close to that for protection of activity (270 nM). The other sulfoxides showed ED₅₀-values for inhibition of 11β-hydroxylase that were substantially higher than the ED₅₀-values for protection. Sulfoxides may have a mixed mode of action in protection of 11β-hydroxylase activity, as previously shown for phenols; they may protect by radical scavenging, but may also need to bind close to the active site of the enzyme where destructive radicals may be formed.     

Histoenzymorphology of β-D-glucuronidase in cortical granules in oocytes of some representative vertebrates

Summary Histoenzymorphologic investigations on -D-glucuronidase in cortical granules in oocytes of some representative vertebrates revealed interesting differences in localisation of the enzyme in the two techniques employed using 8-hydroxyquinoline -D-glucosiduronic acid and naphthol AS-BI -D-glucosiduronic acid as substrates. In the former a nonspecific localisation in the entire cytoplasm of the oocytes excepting nucleus was evident whereas in the latter the cortical granules alone exhibited intense activity. To some extent this difference could be accounted for on the basis of yolk content of the eggs. -D-glucoronidase-rich lysosomal cortical granules were found in the amphibian, reptilian and avian oocytes whereas mammalian oocyte cortical granules were devoid of this enzyme. -D-glucuronidase in cortical granules was sensitive to low concentrations of Hg⁺⁺, Cu⁺⁺, glucuronic and mucic acids. Other histochemical evidence for the lysosomal nature of the cortical granules is presented and discussed. -D-glucuronidase seems to have a role in ovum-nutrition and activation which, thus, make a new addition to the existing knowledge of physiological role of -D-glucuronidase and lysosomes.Dr. A. T. Varute — Research Project guide.     

Conservation of Orchids in China

China,as one of the most important Range States of orchids in the world, has about 1300 species with abundant varieties of orchids. However, owing to over, abuse harvest and illegal trades, the wild populations of these species have declined dramatically, even bringing some species to the edge of extinction. In response to such challenges, the Chinese     

Alteration in virulence characteristics of biofilm cells of Pseudomonas aeruginosa in presence of Tamm-Horsfall protein

Summary Tamm-Horsfall glycoprotein is the most abundant protein in human urine. The present investigation was planned to study the effect of Tamm-Horsfall protein (THP) on elaboration of virulence factors by biofilm cells of Pseudomonas aeruginosa. It was observed that with increase in concentration of THP from 10 to 50 μg/ml there was significant enhancement in elaboration of all the virulence factors by biofilm cells of P. aeruginosa. However, with further increase in concentration of THP from 50 to 70 μg/ml, significant decrease in elaboration of all the virulence traits was observed. Implications of these findings in relation to urinary tract infections caused by P. aeruginosa have been discussed.     

Conservation of Orchids in China

China, as one of the most important Range States of orchids in the world, has about 1300 species withabundant varieties of orchids. However, owing to over, abuse harvest and illegal trades, the wild populations ofthese species have declined dramatically, even bringing some species to the edge of extinction. In response to such challenges, the Chinese government has put conservation of orchids as a priority agenda, taking a series of Innovative and effective measures to protect the orchids in the wild.[第一段]     

Glycosylation ofα 1 glycoprotein in septic shock: Changes in degree of branching and in expression of sialyl Lewisx groups

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of ₁-acid glycoprotein (AGP) was studied in the sera of patients surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A andAleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewis^x (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.Abbreviations AAL Aleuria aurantia lectin - AGP ₁-acid glycoprotein - CAIE crossed affinoimmunoelectrophoresis - Con A Concanavalin A - HSPC human serum protein calibrator - IL-1 interleukin 1 - IL-6 interleukin 6 - LIF leukaemia inhibitory factor - LPS lipopolysaccharide - SLeX sialyl Lewis^x - TNF tumour necrosis factor     

Roles of β-catenin in somitogenesis in rat embryos

Summary We studied the roles of β-catenin in somitogenesis using immunostaining and antisense experiments in rat embryos. High levels of β-catenin appeared transiently in the developing rat somites. Initially, β-catenin accumulation was observed in the core cells of presomitic cell aggregates and then in the lumen of epithelial vesicles. Subsequently, it was confined to the dermomyotomes and their lumen and then the myotomes. High levels of cyclin D1 were observed in the core cells, in the lumen of epithelial vesicles, in myotomes, and in mesenchymal sclerotomes. When embryos were cultured in medium supplemented with β-catenin antisense oligodeoxynucleotide (ODN), the accumulation of β-catenin, but not of cyclin D1, in the nascent somites and dermomyotomes was suppressed, while the number of somites was the same as that observed in control embryos. The number of myosin-positive somites and the amount of myosin per somite in embryos treated with the antisense ODN were lower than those in controls. These results suggested that β-catenin promotes development of myotomal cells during somitogenesis. The function of β-catenin in the development of myotomes may not be correlated to cyclin D1.     

Involvement of GSK-3β in attenuation of the cardioprotective effect of ischemic preconditioning in diabetic rat heart

ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.     

Gene expression of proteins of the vesicle cycle in dopaminergic neurons in modeling of Parkinson’s disease

It is assumed that one of the causes of the degeneration of dopaminergic neurons is the dysregulation of the vesicle cycle, which is ensured by a number of proteins including syntaxin I, synaptotagmin I, complexins I and II, and Rab5. It was shown that there is a compensatory increase in gene expression of proteins responsible for exocytosis at the preclinical stage of Parkinson’s disease (PD) in the in substantia nigra (SN) in mice. Conversely, in the model of the clinical stage of PD, the decreases of gene expression of proteins responsible for exocytosis, endocytosis, and neuronal survival, which may be among the triggers of motor dysfunctions.     

Application of cyclodextrinase in non-complexant production of γ-cyclodextrin

The production of γ-cyclodextrin usually includes the utilization of organic complexants. However, the non-complexant production of γ-cyclodextrin is always being explored due to the defects of organic complexants. However, in non-complexant production, the separation of γ-cyclodextrin from α- and β-cyclodextrin is still a challenge. Here, the selective hydrolysis ability of a cyclodextrinase designated PpCD (cyclodextrinase from Palaeococcus pacificus) on α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin was proved. The k_cat/K_m values of PpCD for α-cyclodextrin and β-cyclodextrin were roughly 12-fold and 5-fold higher than that of γ-cyclodextrin. It was proved that PpCD had selective hydrolysis ability and its γ-cyclodextrin purification performance was apparent on various simulated cyclodextrin mixtures with reported proportions derived from different CGTases. Besides, the hydrolysis temperature was optimized and it could be seen that 85°C was appropriate for the production of γ-cyclodextrin. In addition, the production of γ-cyclodextrin was achieved by using PpCD in the γ-CGTase reaction products.     

Cyclization of lycopene in the biosynthesis of β-carotene

Mycobacterium marinum produces carotenoids when exposed to light or when antimycin A is added. Although the major pigment synthesized is β-carotene, lycopene is accumulated when the induced bacteria are incubated in the presence of nicotine (5 mM) or 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) (50 μM). Both of these compounds inhibit β-carotene synthesis by blocking the cyclization of lycopene. When nicotine is removed by washing the cells, the accumulated lycopene is cyclized to form β-carotene. The cyclization of lycopene is not an energy-requiring reaction and, furthermore, does not require oxygen or any other electron acceptor. Chloramphenicol addition also does not inhibit the conversion of lycopene to β-carotene indicating that no de novo protein synthesis is involved. Nicotine appears to act by inhibiting the activity of the enzyme required for the cyclization of lycopene.Although the mode of action of CPTA is similar to nicotine, it cannot be removed by washing once the cells have been incubated in its presence, suggesting that the molecule is tightly bound to the enzyme. The possible active molecular sites of nicotine and CPTA are discussed.     

Mass spectrometrical analysis of phosphoprotein enriched in astrocytes of 15 kDa in mouse hippocampi

Summary. Phosphoprotein enriched in astrocytes of 15kDa (PEA-15) is a small protein that was first identified as an abundant phosphoprotein in brain. PEA-15 was characterised so far at the immunochemical level and by a microsequencing attempt. In order to update characterisation of this important structure by advanced methodology unambiguously identifying proteins independent of antibody availability and specificity, we used a proteomic method for this purpose: Performing protein profiling in mouse hippocampi using two dimensional gel electrophoresis with subsequent mass spectrometrical (MS/MS) identification we detected this protein and demonstrate proteomic characterisation of PEA-15 (Q62048). This study enables further specific and unambiguous determination serving as an analytical tool.     

The control of hepatic glycogen metabolism in an in vitro model of sepsis

Culturing hepatocytes with a combination of LPS, TNF-α, IL-1β and IFN-γ resulted in an inhibition of glucose output from glycogen and prevented the repletion of glycogen in freshly cultured cells. The reduced glycogen mobilisation correlated with the lower cell glycogen content and reduced rate of glycogen synthesis from [U-¹⁴C]glucose rather than alterations in either total phosphorylase or phosphorylase a activity. There was no change in the percentage of glycogen exported as glucose nor the production of lactate plus pyruvate indicating that redistribution of the Gluc-6-P cannot explain the failure of the liver to export glucose. Although changes in glycogen mobilisation correlated with NO production, inhibition of NO synthase by inclusion of L-NMMA in the culture medium failed to prevent the inhibition of either glycogen accumulation or mobilisation by the proinflammatory cytokines, precluding the involvement of NO in this response. LPS plus cytokine treatment had no effect on total glycogen synthase activity although the activity ratio was lowered, indicative of increased phosphorylation. The inhibition of glycogen synthesis correlated with a fall in the intracellular concentrations of Gluc-6-P and UDP-glucose and in the absence of measured changes in kinase activity, it is suggested that the fall in Gluc-6-P reduces both substrate supply and glycogen synthase phosphatase activity. The fall in Gluc-6-P coincided with a reduction in total glucokinase and hexokinase activity within the cells, but no significant change in either the translocation of glucokinase or glucose-6-phosphatase activity. This demonstrates direct cytokine effects on glycogen metabolism independent of changes in glucoregulatory hormones.