Crystal structure of amylose complexes with small ligands

Amylose complexes were prepared, as lamellar single crystals and polycrystalline powders, from aqueous solutions by adding small flavor molecules. The morphology, crystal structure, and thermal properties of complexes with fenchone, menthone, and geraniol were determined using transmission electron microscopy, wide-angle X-ray diffraction, and differential scanning calorimetry. The crystal structure was found to be similar to that of V amylose complexes with isopropanol. This implies that the crystallosolvates contain sixfold helices packed in orthorhombic unit cells, with the ligands possibly lying in the interhelical space. Different drying procedures were also studied leading to less resolved X-ray diagrams. The thermoanalysis confirmed that complexes with a relatively high crystallinity were formed.     

Microbial starch-binding domains as a tool for targeting proteins to granules during starch biosynthesis

Modification of starch biosynthesis pathways holds an enormous potential for tailoring granules or polymers with new functionalities. In this study, we explored the possibility of engineering artificial granule-bound proteins, which can be incorporated in the granule during biosynthesis. The starch-binding domain (SBD)-encoding region of cyclodextrin glycosyltransferase from Bacillus circulans was fused to the sequence encoding the transit peptide (amyloplast entry) of potato granule-bound starch synthase I (GBSS I). The synthetic gene was expressed in the tubers of two potato cultivars (cv. Kardal and cv. Karnico) and one amylose-free (amf) potato mutant. SBDs accumulated inside starch granules, not at the granule surface. Amylose-free granules contained 8 times more SBD (estimated at ca. 1.6% of dry weight) than the amylose-containing ones. No consistent differences in physicochemical properties between transgenic SBD starches and their corresponding controls were found, suggesting that SBD can be used as an anchor for effector proteins without having side-effects. To test this, a construct harbouring the GBSS I transit peptide, the luciferase reporter gene, a PT-linker, and the SBD (in frame), and a similar construct without the linker and the SBD, were introduced in cv. Kardal. The fusion protein accumulated in starch granules (with retainment of luciferase activity), whereas the luciferase alone did not. Our results demonstrate that SBD technology can be developed into a true platform technology, in which SBDs can be fused to a large choice of effector proteins to generate potato starches with new or improved functionalities.     

Prevalence of coccidia parasites (Protozoa) in red squirrels ( Sciurus vulgaris): effects of host phenotype and environmental factors

We investigated the relative importance of environmental factors versus host phenotype in determining parasite prevalence in Eurasian red squirrels ( Sciurus vulgaris). One hundred and forty-three fecal samples of 116 different squirrels collected in 2000 and 2001 from five study areas in the Italian Alps, were examined for intestinal protozoans. Two species of Eimeria were present with a medium to high prevalence in both years and in all areas, while two other species were rare, occurring only in some areas and not in all years. Cryptosporidium parvum had a high prevalence in the two study areas of the Western Alps, while in the three areas of the Central Alps it was recorded only once. The prevalence of Eimeria sciurorum and C. parvum fluctuated in parallel with squirrel density, suggesting a possible correlation between the presence of these protozoans and host density. A gender effect on E. sciurorum prevalence at low density could be explained by different space use patterns and social organization of males and females. C. parvum occurred more frequently in young squirrels, suggesting an acquired immunity in adults, but age-related susceptibility was not found for eimerian species. The coccidian community was more similar within than between regions, and study area and year were key parameters in predicting coccidia infection. There was no evidence of competition between coccidian species, but one positive interaction between E. sciurorum and E. andrewsi was observed. Our results suggest that the effects of geographic region, area features, and year effects probably related to fluctuations in host population density, were more important than individual phenotypic host characteristics in structuring the coccidian assemblage and determining levels of parasite prevalence in red squirrel populations.     

Sex steroid hormones do not influence the oxidative burst activity of polymorphonuclear leukocytes from ovariectomized cows in vitro

During the periparturient period, dairy cows are subjected to physiological changes that may induce immunosuppression and an increased susceptibility of the animal to bacterial infections such as mastitis. The incidence of clinical environmental mastitis is high during the last period of gestation, at parturition and during the first month of lactation, suggesting a potential influence of sex steroid hormones. Efficient functioning of polymorphonuclear leukocytes (PMN) is necessary during the early phase of infection to clear the mammary gland from invading pathogens. The purpose of this study was to investigate the effect of sex steroid hormones on the oxidative burst activity of isolated PMN from ovariectomized cows. Ovariectomy was performed to minimize the interference of endogenous estrogen and progesterone levels, which are known to vary extensively during the estrus cycle. Isolated PMN were incubated with different concentrations of 17beta-estradiol, estrone or progesterone. A flow cytometric technique was used to quantify the oxidation of intracellular 2',7'-dichlorofluorescin by the oxidative burst system of PMN following stimulation with phorbol myristate acetate. Staurosporine was used as a positive control for our in vitro model. No statistically significant changes in PMN oxidative burst activity were observed at physiological or pharmacological levels of the three sex steroid hormones. A large variation existed in the oxidative burst activity among cows. In an additional experiment, the expression of estrogen receptor alpha and of progesterone receptor in PMN was evaluated immunohistochemically. No specific staining was detected for both receptors in isolated PMN following incubation with different concentrations of sex steroid hormones.     

Myenteric nitrergic neurons along the rat esophagus: evidence for regional and strain differences in age-related changes

Several studies have suggested an age-related reduction in the number of myenteric neurons in the lower gastrointestinal (GI) tract linked to changes in GI neuromuscular functions. The present study, combining protein gene product 9.5 immunostaining and NADPH-diaphorase histochemistry, aimed at quantifying the proportion of nitrergic neurons compared to the overall number of enteric neurons in the esophagus of young (3-4.5 months) and aged (18-20 months) Sprague-Dawley and Wistar rats. In both strains, the neuron numbers per ganglion in the cervical region were almost twice as high as in the other esophageal regions. Irrespective of age or strain, the esophagus harbored a very high proportion of intrinsic nitrergic neurons (greater than approximately 65%). Both strains showed with aging an overall neuronal loss of approximately 27%. While a significant increase (young: 64-71%; aged: 82-89%) was observed in all esophageal regions in the Wistar strain, the proportion of nitrergic neurons remained stable with aging in the Sprague-Dawley strain (range: 72-82%). In conclusion, the age-related reduction in the overall number of myenteric, nitrergic, and non-nitrergic neurons observed in the rat esophagus, appears to be highly region- and strain-dependent. Therefore, a protective mechanism against neuronal cell loss, selectively present in specific (nitrergic) enteric subpopulations, as suggested in earlier reports, cannot be put forward as a general phenomenon throughout the entire GI tract.     

The WFS1 gene,responsible for low frequency sensorineural hearing loss and Wolfram syndrome,is expressed in a variety of inner ear cells

Heterozygous mutations in the WFS1 gene are responsible for autosomal dominant low frequency hearing loss at the DFNA6/14 locus, while homozygous or compound heterozygous mutations underlie Wolfram syndrome. In this study we examine expression of wolframin, the WFS1-gene product, in mouse inner ear at different developmental stages using immunohistochemistry and in situ hybridization. Both techniques showed compatible results and indicated a clear expression in different cell types of the inner ear. Although there were observable developmental differences, no differences in staining pattern or gradients of expression were observed between the basal and apical parts of the cochlea. Double immunostaining with an endoplasmic reticulum marker confirmed that wolframin localizes to this organelle. A remarkable similarity was observed between cells expressing wolframin and the presence of canalicular reticulum, a specialized form of endoplasmic reticulum. The canalicular reticulum is believed to be involved in the transcellular movements of ions, an important process in the physiology of the inner ear. Although there is nothing currently known about the function of wolframin, our results suggest that it may play a role in inner ear ion homeostasis as maintained by the canalicular reticulum.     

Anti-beta2-glycoprotein I antibodies recognizing platelet factor 4-heparin complex in antiphospholipid syndrome in patient substantiated with mouse model

The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies associated with arterial and/or venous thrombosis, and recurrent abortion accompanied often by thrombocytopenia. These antibodies are heterogeneous and react against phospholipid-binding proteins such as beta2-glycoprotein I (beta2GPI) and prothrombin. The recognition of anti-beta2-glycoprotein I (anti-beta2GPI) by platelet factor 4-heparin complex (PF4-Hc) has been previously evoked and partially confirmed by the present inhibition studies. Further, the anti-beta2-glycoprotein I antibodies were purified from a patient with primary antiphospholipid syndrome using Affi-gel-10-beta2GPI immunoaffinity chromatography. The purified anti-beta2GPI IgM as well as patient serum equally recognized PF4-Hc in ELISA mode. In order to substantiate this data and to better understand we studied an animal model using mouse active immunization with the purified human anti-beta2GPI. The mice showed a significant decrease in their platelet count. In addition the ELISA responses of the immunized mice sera were positive against both beta2GPI and PF4-Hc, substantiating the double recognition. Despite many previous reported animal model studies, this is the first time we have shown the specific recognition of anti-beta2GPI antibodies by PF4-Hc, the results in the induced mice correlating the data observed with some patients.     

Quantification of tagitinin C in Tithonia diversifolia by reversed-phase high-performance liquid chromatography

A simple, rapid and reliable reversed-phase high-performance liquid chromatographic method for the determination of tagitinin C, an anti-plasmodial sesquiterpene lactone isolated from the aerial parts of Tithonia diversifolia, has been developed. The assay has been used to quantify tagitinin C in various extracts of the aerial parts of T. diversifolia.     

Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.