Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize (Zea mays) and pearl millet (Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high -helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, ¹H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded -helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.     

CD69 expression induced by thapsigargin,phorbol ester and ouabain on thymocytes is dependent on external Ca2+ entry

In the present work murine thymocytes exposed to Thapsigargin (TG 10, 20 and 50 nM), Phorbol-12,13,20-triacetate (TPA16 nM) and Ouabain (OUA100 nM) exhibited an increased expression of CD69, a molecule related to cellular activation and associated to Ca(++) influx in other systems. The kinetics of CD69 appearance depended on the stimuli and dose used. TG 50 nM induced an increased expression by 6 h whereas with lower doses (10 and 20 nM) an increase was detected at 18 h. TPA maximal increase was evident at 6 h. OUA lead to an observable increase at 18 h. However, in the case of TPA or TG the presence of the stimuli was only necessary for the first 2 h of culture, whereas OUA needed to be present during the whole assay. It was also demonstrated that Ca(++) influx was an essential feature, as EGTA diminished or abolished CD69 increased expression. Nevertheless, EGTA was only capable of this effect when present at the time of the stimuli. No correlation of CD69 expression with thymocyte death was observed. Similarly, the agents under study did not promote the maturation from double-positive into single-positive thymocytes. TPA and Thapsigargin were capable of decreasing the level of CD4 molecules on the cell surface, probably due to the loss of these molecules. OUA, on the other hand, did not modify CD4/CD8 expression on these cells.     

Hereditary transthyretin amyloidosis: molecular basis and therapeutical strategies

Transthyretin (TTR) is a transport protein for thyroid hormones and vitamin A and might have an important role in the nervous system. However, TTR can undergo a conformational change and form amyloid fibrils, in both acquired and hereditary forms of systemic amyloidosis. More than 80 TTR mutations have been associated with autosomal dominant amyloidosis, usually presenting with peripheral and autonomic neuropathy and/or cardiomyopathy. Major areas of research in TTR amyloidosis include: molecular mechanisms leading to fibril formation; mechanisms of fibril-induced cell death; modulators of phenotypic expression of the disease; and therapeutic strategies.     

A prokaryotic condensin/cohesin-like complex can actively compact chromosomes from a single position on the nucleoid and binds to DNA as a ring-like structure

We show that Bacillus subtilis SMC (structural maintenance of chromosome protein) localizes to discrete foci in a cell cycle-dependent manner. Early in the cell cycle, SMC moves from the middle of the cell toward opposite cell poles in a rapid and dynamic manner and appears to interact with different regions on the chromosomes during the cell cycle. SMC colocalizes with its interacting partners, ScpA and ScpB, and the specific localization of SMC depends on both Scp proteins, showing that all three components of the SMC complex are required for proper localization. Cytological and biochemical experiments showed that dimeric ScpB stabilized the binding of ScpA to the SMC head domains. Purified SMC showed nonspecific binding to double-stranded DNA, independent of Scp proteins or ATP, and was retained on DNA after binding to closed DNA but not to linear DNA. The SMC head domains and hinge region did not show strong DNA binding activity, suggesting that the coiled-coil regions in SMC mediate an association with DNA and that SMC binds to DNA as a ring-like structure. The overproduction of SMC resulted in global chromosome compaction, while SMC was largely retained in bipolar foci, suggesting that the SMC complex forms condensation centers that actively affect global chromosome compaction from a defined position on the nucleoid.     

Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family

Human intestinal cells lack globotriaosylceramide (Gb(3)), the receptor for Shiga toxin-1 (Stx1) and Shiga toxin-2 (Stx2). Therefore, the role of these toxins in mediating intestinal disease during infection with Shiga toxin-producing Escherichia coli is unclear. The aims of this study were to determine whether Stx1 and Stx2 induce apoptosis in epithelial cells expressing (HEp-2, Caco-2) or lacking (T84) Gb(3) and to characterize the role of the Bcl-2 family. Stx1 (12.5 ng/ml) induced apoptosis in both HEp-2 (21.9 +/- 7.9% vs. 0.8 +/- 0.3%, P = 0.01) and Caco-2 (10.1 +/- 1.2% vs. 3.1 +/- 0.4%, P = 0.006) cells but not in Gb(3)-deficient T84 cells. Toxin-mediated apoptosis of HEp-2 cells was associated with enhanced expression of the proapoptotic protein Bax. Inhibition of caspase activation prevented toxin-stimulated apoptosis. In addition, overexpression of Bcl-2 by transient transfection blocked Stx1-stimulated cell death. These findings indicate that Shiga toxins produced by E. coli signal Gb(3)-expressing epithelial cells to undergo apoptosis in association with enhanced Bax expression, thereby resulting in activation of the caspase cascade.     

MEI1, an Arabidopsis gene required for male meiosis: isolation and characterization

 The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces after meiosis an abnormal tetrad, consisting of five to eight microspores of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-type genomic library, using the plant flanking DNA as a probe. Using RT-PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic sequence in this region indicated a gene which contained two introns. The 5′ and 3′ splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. The deduced sequence of the MEI1 wild-type gene, which contains 89 amino acids, shows possible similarity with the human acrosin-trypsin inhibitor, HUSI-II, and is about the same size. Two wild-type DNA fragments, both extending over the T-DNA insertion site, were introduced into mutant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several independent male-fertile transformants were obtained with one of the DNA fragments. The fragment showing complementation of the mutant phenotype indicated that the sequence with similarity to the acrosin-trypsin inhibitor is MEI1. Within the 16-kb genomic fragment two other genes were identified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Arabidopsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characterization of the mei1 phenotype because the individual microspores continued to be held together after callose dissolution. Received: 21 April 1998 / Revision accepted: 11 June 1998     

Anthelmintic Activity In Vivo of Epiisopiloturine against Juvenile and Adult Worms of Schistosoma mansoni

Schistosomiasis is a serious disease currently estimated to affect more that 207 million people worldwide. Due to the intensive use of praziquantel, there is increasing concern about the development of drug-resistant strains. Therefore, it is necessary to search for and investigate new potential schistosomicidal compounds. This work reports the in vivo effect of the alkaloid epiisopiloturine (EPI) against adults and juvenile worms of Schistosoma mansoni. EPI was first purified its thermal behavior and theoretical solubility parameters charaterised. In the experiment, mice were treated with EPI over the 21 days post-infection with the doses of 40 and 200 mg/kg, and 45 days post-infection with single doses of 40, 100 and 300 mg/kg. The treatment with EPI at 40 mg/kg was more effective in adult worms when compared with doses of 100 and 300 mg/kg. The treatment with 40 mg/kg in adult worms reduced parasite burden significantly, lead to reduction in hepatosplenomegaly, reduced the egg burden in faeces, and decreased granuloma diameter. Scanning electron microscopy revealed morphological changes to the parasite tegument after treatment, including the loss of important features. Additionally, the in vivo treatment against juvenile with 40 mg/kg showed a reduction of the total worm burden of 50.2%. Histopathological studies were performed on liver, spleen, lung, kidney and brain and EPI was shown to have a DL50 of 8000 mg/kg. Therefore EPI shows potential to be used in schistosomiasis treatment. This is the first time that schistosomicidal in vivo activity of EPI has been reported.     

Experimental study and constitutive modeling of the viscoelastic mechanical properties of the human prolapsed vaginal tissue

In this paper, the viscoelastic mechanical properties of vaginal tissue are investigated. Using previous results of the authors on the mechanical properties of biological soft tissues and newly experimental data from uniaxial tension tests, a new model for the viscoelastic mechanical properties of the human vaginal tissue is proposed. The structural model seems to be sufficiently accurate to guarantee its application to prediction of reliable stress distributions, and is suitable for finite element computations. The obtained results may be helpful in the design of surgical procedures with autologous tissue or prostheses.     

Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species

Background and Aims

Intraspecific ploidy-level variation is an important aspect of a species'' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species.

Methods

Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome.

Key Results

Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north–south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names.

Conclusions

Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.