Carbonate dehydratase (carbonic anhydrase) in a spider. Association with the hemolymph lipoprotein

Carbonate dehydratase was detected dissolved in the hemolymph of the tarantula, Eurypelma californicum. The enzyme was purified 31-fold by gel filtration, anion-exchange chromatography, a second gel filtration, and finally, preparative polyacrylamide gel electrophoresis. Zinc content increased during purification to up to 2.4 mol Zn/100 000 g of protein (= 1.58 mg Zn/g protein). In the polyacrylamide electrophoresis of tarantula hemolymph under non-denaturing conditions three major protein bands were observed: hemocyanin, a 16 S lipoprotein and the active band which migrated closely behind the 16 S lipoprotein. After treatment with sodium dodecyl sulfate both the carbonate dehydratase-active protein and the lipoprotein revealed bands corresponding to Mr = 95 000 and 110 000, respectively, but the enzymatically active protein revealed an additional third band with Mr = 40 000. The latter band is though to represent the 'true' carbonate dehydratase protein. Upon isoelectric focusing of material containing carbonate dehydratase activity and lipoprotein, bands were obtained at pH 5.45, 5.6 and 5.7. The band at pH 5.6 contained the peak of enzyme activity, and upon dodecyl sulfate-polyacrylamide gel electrophoresis showed the highest proportion of the 40-kDa polypeptide. It is concluded that tarantula carbonate dehydratase, instead of forming a high molecular mass aggregate, is associated with the 16 S lipoprotein, the latter serving as a carrier for the enzyme. The lipoprotein is probably also involved in other transport processes. It is present in great excess and may therefore occur in two forms, charged with carbonate dehydratase or uncharged. Tarantula carbonate dehydratase is inhibited by acetazolamide and by dansylamide, but not by a number of other known inhibitors, most notably not by 4-(aminomethyl)benzenesulfonamide. Treatment with 1M urea does not affect specific enzyme activity, while 2M urea inhibits by 50%. 2-Mercaptoethanol inhibits activity by 50% at 0.1M. Like other carbonate dehydratases, the tarantula enzyme shows esterase activity. The Km for 4-nitrophenyl acetate is 5mM.     

Der Tryptophanstoffwechsel und die Ommochromsynthese während der Embryonalentwicklung der Stabheuschrecke Carausius morosus

The synthesis of ommochromes, ommin, and xanthommatin, was studied during the embryonic development of Carausius morosus. Ommochrome synthesis begins in the epidermis of the embryo at the 46th day after egg laying. The ommochrome accumulation is directly related to the continuous increase of pigment spots on the epidermis of the pharate larva.The concentration of free tryptophan increases progressively from the germ band formation (15th day), until the final position of the embryo in the egg, with the head immediately beneath the operculum (44th day). Subsequent decrease in concentration of tryptophan coincides with the appearance of ommochrome in the epidermis of the embryo. The tryptophan levels decrease considerably from 44th day till the 60th day. During the stage of the pharate first instar larva tryptophan levels remain low until hatching. The concentrations of the intermediates, kynurenine and 3-HO-kynurenine, show no accumulation and remain low during the whole of ommochrome synthesis.     

Mycorrhizal suppression and phosphorus addition influence the stability of plant community composition and function in a temperate steppe

Nutrient enrichment can reduce ecosystem stability, typically measured as temporal stability of a single function, e.g. plant productivity. Moreover, nutrient enrichment can alter plant–soil interactions (e.g. mycorrhizal symbiosis) that determine plant community composition and productivity. Thus, it is likely that nutrient enrichment and interactions between plants and their soil communities co-determine the stability in plant community composition and productivity. Yet our understanding as to how nutrient enrichment affects multiple facets of ecosystem stability, such as functional and compositional stability, and the role of above–belowground interactions are still lacking. We tested how mycorrhizal suppression and phosphorus (P) addition influenced multiple facets of ecosystem stability in a three-year field study in a temperate steppe. Here we focused on the functional and compositional stability of plant community; functional stability is the temporal community variance in primary productivity; compositional stability is represented by compositional resistance, turnover, species extinction and invasion. Community variance was partitioned into population variance defined as community productivity weighted average of the species temporal variance in performance, and species synchrony defined as the degree of temporal positive covariation among species. Compared to treatments with mycorrhizal suppression, the intact AM fungal communities reduced community variance in primary productivity by reducing species synchrony at high levels of P addition. Species synchrony and population variance were linearly associated with community variance with the intact AM fungal communities, while these relationships were decoupled or weakened by mycorrhizal suppression. The intact AM fungal communities promoted the compositional resistance of plant communities by reducing compositional turnover, but this effect was suppressed by P addition. P addition increased the number of species extinctions and thus promoted compositional turnover. Our study shows P addition and AM fungal communities can jointly and independently modify the various components of ecosystem stability in terms of plant community productivity and composition.     

Inhibition of intestinal ischemia/repurfusion induced apoptosis and necrosis via down-regulation of the NF-kB,c-Jun and caspace-3 expression by epigallocatechin-3-gallate administration

Intestinal ischemia/reperfusion (I/R) produces reactive oxygen species (ROS) activating signal transduction and apoptosis. The aim of this study was to evaluate the effect of (?)-epigallocatechin-3-gallate (EGCG) administration in inhibition of apoptosis by attenuating the expression of NF-kB, c-Jun and caspace-3 in intestinal I/R. Thirty male wistar rats were used. Group A sham operation, B I/R, C I/R-EGCG 50 mg/kg ip. Intestinal ischemia was induced for 60 min by clamping the superior mesenteric artery. Malondialdehyde (MDA), myeloperoxidase (MPO), light histology, Fragment End Labelling of DNA (TUNEL), immunocytochemistry for NF-kB, c-Jun and caspace-3 analysis in intestinal specimens were performed 120 min after reperfusion. Apoptosis as indicated by TUNEL and Caspace-3, NF-kB and c-Jun was widely expressed in I/R group but only slightly expressed in EGCG treated groups. MDA and MPO showed a marked increase in the I/R group and a significant decrease in the EGCG treated group. Light histology showed preservation of architecture in the EGCG treated group. In conclusion, EGCG pre-treatment is likely to inhibit intestinal I/R-induced apoptosis by down-regulating the expression of NF-kB, c-Jun and caspase-3.     

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.     

Interplay between apoptotic and autophagy pathways after exposure to cerium dioxide nanoparticles in human monocytes

Engineered nanomaterials, defined as having at least one dimension smaller than 100 nm, have revolutionized many technology sectors ranging from therapeutics and diagnostics to environmental monitoring and remediation. This has resulted in a rapid increase in their manufacture over the past few years, accompanied by an increased human exposure potential. However, understanding of the interactions of nanomaterials with biological systems is still rudimentary. We have described that an environmentally and medically relevant nano metal (cerium dioxide) can affect primary human monocyte viability and interact with programmed cell death pathways leading to apoptosis and autophagic cell death. Cerium dioxide nanoparticles (CeO₂ NPs)-induced autophagy acts as a prodeath mechanism and leads to increased cytotoxicity of human monocytes. A better understanding of the implication and biological significance of CeO₂ NPs-induced autophagy and apoptosis will help us understand the risks associated with its uses and develop safer nanomedicine.     

Radiochemical and radiobiological assessment of a pyridyl-S-cysteine functionalized bombesin derivative labeled with the 99mTc(CO)3+ core

Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2′-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH₂) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)₃(H₂O)₃]⁺ (M = ^99mTc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new ^99mTc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images.     

Quinidine: an “Endangered Species” Drug Appropriate for Management of Electrical Storm in Brugada Syndrome

Brugada syndrome is an inherited channelopathy associated with an increased risk of syncope and sudden cardiac death. In rare cases it can be manifested with electrical storm. We report two cases of Brugada syndrome that presented with electrical storm and were treated successfully with oral quinidine, an "endangered species" drug.     

Effect of Acute Negative and Positive Energy Balance on Basal Very-Low Density Lipoprotein Triglyceride Metabolism in Women

Background

Acute reduction in dietary energy intake reduces very low-density lipoprotein triglyceride (VLDL-TG) concentration. Although chronic dietary energy surplus and obesity are associated with hypertriglyceridemia, the effect of acute overfeeding on VLDL-TG metabolism is not known.

Objective

The aim of the present study was to investigate the effects of acute negative and positive energy balance on VLDL-TG metabolism in healthy women.

Design

Ten healthy women (age: 22.0±2.9 years, BMI: 21.2±1.3 kg/m²) underwent a stable isotopically labeled tracer infusion study to determine basal VLDL-TG kinetics after performing, in random order, three experimental trials on the previous day: i) isocaloric feeding (control) ii) hypocaloric feeding with a dietary energy restriction of 2.89±0.42 MJ and iii) hypercaloric feeding with a dietary energy surplus of 2.91±0.32 MJ. The three diets had the same macronutrient composition.

Results

Fasting plasma VLDL-TG concentrations decreased by ∼26% after hypocaloric feeding relative to the control trial (P = 0.037), owing to decreased hepatic VLDL-TG secretion rate (by 21%, P = 0.023) and increased VLDL-TG plasma clearance rate (by ∼12%, P = 0.016). Hypercaloric feeding increased plasma glucose concentration (P = 0.042) but had no effect on VLDL-TG concentration and kinetics compared to the control trial.

Conclusion

Acute dietary energy deficit (∼3MJ) leads to hypotriglyceridemia via a combination of decreased hepatic VLDL-TG secretion and increased VLDL-TG clearance. On the other hand, acute dietary energy surplus (∼3MJ) does not affect basal VLDL-TG metabolism but disrupts glucose homeostasis in healthy women.