Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm

Bull sperm were treated with several levels of cholesterol-loaded cyclodextrin (CLC) and frozen in egg yolk diluents containing either Tris or sodium citrate, to determine the CLC concentration that best benefits bull sperm cryosurvival. After thawing, higher percentages of motile (60%) and viable (55%) sperm were obtained when 1.5mg/ml CLC was added to sperm prior to freezing, than for sperm frozen in egg yolk Tris alone (42 and 46%, respectively; P < 0.05). Increasing concentration of CLCs, maintained higher percentages of viable sperm up to addition of 6.0mg/ml CLC when the percentages of viable sperm began to decline (50%; P < 0.05). Addition of 1.5mg/ml CLC to sperm frozen in sodium citrate diluent resulted in 53% motile sperm compared to 37% for control, although these were not different (P > 0.05). The beneficial effects of CLC addition were observed regardless of whether sperm incubated with CLC at 22 or 37 degrees C (P > 0.05) and maximum effects were observed when sperm incubated with CLC for 15min. Longer incubation times, up to 60min, resulted in similar results (P > 0.05). The amount of cholesterol that incorporated into sperm, increased with increasing CLC concentration, in a linear fashion, and each sperm incorporates a similar amount of cholesterol (coefficient of variation=12.9+/-0.7%). In addition, the cholesterol incorporates into all sperm membranes. Increasing membrane cholesterol levels, by adding CLCs to cells, prior to freezing, is a simple technology that increases the cryosurvival of bull sperm, and may benefit the cryosurvival of many cell types.     

Cyclodextrin-bound 6-(4-methoxyphenyl)imidazo[1,2-alpha+/-]pyrazin-3(7H)-ones with fluorescein as green chemiluminescent probes for superoxide anions

In providing chemiluminescent probes that have high chemiluminescence intensity and high specificity to superoxide anions, novel chemiluminescent probes involving cyclodextrins covalently bound to 6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-one with fluorescein were synthesized and characterized. Using the hypoxanthine-xanthine oxidase system for the generation of the superoxide anions, these novel chemiluminescent probes showed higher superoxide-induced chemiluminescence intensity than that of 6-[4-[2-[N(')-(5-fluoresceinyl)thioureido]-ethoxy]phenyl]-2-methylimidazo[1,2-alpha]pyrazin-3(7H)-one (FCLA). When tested at a probe concentration of 1.0 microM, compound 6, in which 6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-one and fluorescein are covalently attached on the secondary and primary hydroxyl faces of gamma-cyclodextrin, respectively, showed green luminescence intensity that was 26 times that of FCLA, which was also the highest luminescence intensity in this present study. At probe concentrations of less than 1.0 microM, the ratio of the superoxide-dependent chemiluminescence intensity to the background chemiluminescence intensity for compound 6 was higher than that of FCLA. This high superoxide-induced chemiluminescence intensity and superoxide specificity in low probe concentrations indicates that 6 can be more effective than FCLA toward the measurement of superoxide anions.     

PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles

In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear beta-cyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.     

Use of cyclodextrin as an agent to induce excretion of Bordetella pertussis antigens

Abstract This paper attempts to provide an explanation for the effect of cyclodextrin on the yield of Bordetella pertussis soluble antigens. It was demonstrated that the addition of cyclodextrin to the synthetic Stainer-Scholte liquid medium enhances the level of the intracellular form of adenylate cyclase (200 kDa) in the supernate. In addition to this effect, it has been reported that cyclodextrin also enhances the levels of two other extracellular proteins, pertussis toxin and filamentous hemagglutinin. As these antigens are structurally different, it seems that the effect of cyclodextrin is not specific. With the use of different buffer systems of well-known action on outer membrane stability it was possible to determine a relationship between the presence of cyclodextrin, destabilisation of the outer membrane and the release of proteins. It was determined that the cyclodextrin did not modify the fluidity of B. pertussis cells but produced a change of outer membrane permeability.     

Insolubilization of exogenous primer dextran with 1,3-α-d-glucan synthase from Streptococcus mutans

Abstract The water-insolubilization mechanism of exogenous primer dextran with 1,3-α- d -glucan synthase (EC 2.4.1.-) from Streptococcus mutans was studied. The 1,3-α- d -glucan synthase solution, containing sucrose and exogenous primer dextran, was incubated briefly. Water-insoluble glucan was synthesized. At the same time, water-soluble glucan, mainly derived from exogenous primer dextran, decreased. Linkage analysis data of glucan produced revealed that 1,3-α- d -glucoside bonds increased. Exogenous primer dextran was changed by the action of 1,3-α- d -glucan synthase to water-insoluble glucan. The results suggest that in a short-term reaction system of outside primer-insertion type, the 1,6-α- d -glucoside bond forms the main chain of water-insoluble glucan.     

A supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities

For constructing a bifunctional antioxidative enzyme with both superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, a supramolecular artificial enzyme was successfully constructed by the self-assembly of the Mn(III)meso-tetra[1-(1-adamantyl methyl ketone)-4-pyridyl] porphyrin (MnTPyP-M-Ad) and cyclodextrin-based telluronic acid (2-CD-TeO₃H) through host-guest interaction in aqueous solution. The self-assembly of the adamantyl moieties of Mn(III) porphyrin and the β-CD cavities of 2-CD-TeO₃H was demonstrated by the NMR spectra. In this supramolecular enzyme model, the Mn(III) porphyrin center acted as an efficient active site of SOD and tellurol moiety endowed GPx activity. The SOD-like activity (IC₅₀) of the new catalyst was found to be 0.116 μM and equals to 2.56% of the activity of the native SOD. Besides this, supramolecular enzyme model also showed a high GPx activity, and a remarkable rate enhancement of 27-fold compared to the well-known GPx mimic ebselen was observed. More importantly, the supramolecular artificial enzyme showed good thermal stability.     

Subcellular localization of flavonoid synthesizing enzymes in Pisum,Phaseolus, Brassica and Spinacia cultivars

Functionally-intact chloroplasts were obtained from 11-day-old pea (Pisum sativum cv Midfreezer) seedlings. Enzyme-distribution studies with ribulose bisphosphate carboxylase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase showed that ca 2.1% of the total tissue chloroplasts were present in the chloroplast preparation. The rate of intactness of chloroplast preparations was 34–82%. SAM:caffeic acid methyltransferase, flavanone synthase, UDPG:flavonoid-3-O-glucosyltransferase and SAM:quercetin methyltransferase activities were measured in the homogenate, supernatant and chloroplast lysate fractions. Significant activities of the above four enzymes could only be detected in the homogenate and supernatant fractions. Similar experiments with 11-day-old seedlings of green beans (Phaseolus vulgaris cv Early Gallatin), red cabbage (Brassica oleracea cv Red Danish) and 6-week-old plants of spinach (Spinacia oleracea cv Bloomsdale) showed a similar distribution of the flavonoid synthesizing enzymes. We conclude that under the reported conditions chloroplasts are not involved in flavonoid biosynthesis.     

Novel insight into the secretory expression of recombinant enzymes in Escherichia coli

The secretory expression of recombinant enzymes in Escherichia coli has generally been a challenging task. In the present study, we investigated the expression of the extracellular enzyme cyclodextrin glycosyltransferase in E. coli. Our results indicated that when the overexpressed pre-proteins were not translocated across the inner membrane in a timely manner, they aggregated near the inner side of the E. coli inner membrane, resulting in the formation of insoluble inclusion bodies, which eventually blocked the pre-protein translocation channels and subsequently impeded further protein secretion. This mechanism suggests that for the efficient production of extracellular enzymes in E. coli, it is very important to maintain a balance between the rate of pre-protein synthesis and translocation, which can be achieved by altering the cultivation process. Our findings provide novel insight into the secretory expression of extracellular enzymes and may shed light on the further development of new strategies for extracellular protein production in E. coli.     

Pathological roles of ceramide and its metabolites in metabolic syndrome and Alzheimer's disease

The public health burden of metabolic syndrome (MetS), a multiplex risk factor that arises from insulin resistance accompanying abnormal adipose conditions, and Alzheimer's disease (AD), the most common form of dementia, continues to expand. Current available therapies for these disorders are of limited effectiveness. Recent findings have indicated that alternations in sphingolipid metabolism contribute to the development of these pathologies. Sphingolipids are major constituents of the plasma membrane, where they are known to form several types of microdomains, and are potent regulators for a variety of physiological processes. Many groups, including ours, have demonstrated that membrane sphingolipids, especially ceramide and its metabolites such as ceramide 1-phosphate, have roles in arteriosclerosis, obesity, diabetes, and inflammation associated with MetS. Aberrant sphingolipid profiles have been observed in human AD brains, and accumulated evidence has demonstrated that changes in membrane properties induced by defective sphingolipid metabolism impair generation and degradation of amyloid-β peptide (Aβ), a pathogenic agent of AD. In this review, we summarize current knowledge and pathophysiological implications of the roles of SLs in MetS and AD, to provide insight into the SL metabolic pathways as potential targets for therapy of these diseases. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.