Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dissociated epithelial cells from Bombyx mori

Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with labelled 125I-Cry1Ab revealed a marked reduction in reversible and irreversible binding when anti-BtR175 (a cadherin-like protein) was used for BBMV pre-treatment. By contrast, the anti-APN1 antibody specifically affected the irreversible binding, while the reversible binding component was not affected. This is the first time that binding of Cry1Ab to APN1 and to a cadherin-like protein from BBMVs in solution has been shown. Dissociated epithelial cells from the Kin strain were used to test the inhibitory effect of the antibodies on the cytotoxicity of Cry1Ab. Pre-incubation of the cells with the anti-BtR175 antibody conferred protection against Cry1Ab, but not the anti-APN1 antibody. Therefore our results seem to support the two models of the mode of action of Cry1Ab in Lepidoptera, depending on whether BBMVs or intact dissociated cells are used, suggesting that both pathways may co-operate for the toxicity of Cry1A toxins in vivo.     

Possible involvement of CD81 in acrosome reaction of sperm in mice

Tetraspanin CD81 is closely homologous in amino acid sequence with CD9. CD9 is well known to be involved in sperm-egg fusion, and CD81 has also been reported to be involved in membrane fusion events. However, the function of CD81 as well as that of CD9 in membrane fusion remains unclear. Here, we report that disruption of the mouse CD81 gene led to a reduction in the fecundity of female mice, and CD81-/- eggs had impaired ability to fuse with sperm. Furthermore, we demonstrated that when CD81-/- eggs were incubated with sperm, some of the sperm that penetrated into the perivitelline space of CD81-/- eggs had not yet undergone the acrosome reaction, indicating that the impaired fusibility of CD81-/- eggs may be in part caused by failure of the acrosome reaction of sperm. In addition, we showed that CD81 was highly expressed in granulosa cells, somatic cells that surround oocytes. Our observations suggest that there is an interaction between sperm and CD81 on somatic cells surrounding eggs before the direct interaction of sperm and eggs. Our results may provide new clues for clarifying the cellular mechanism of the acrosome reaction, which is required for sperm-egg fusion.     

Tektin5, a new Tektin family member, is a component of the middle piece of flagella in rat spermatozoa

Tektins are composed of a family of filament-forming proteins localized in cilia and flagella. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin2 and Tektin4, have been verified to be present in sperm flagella. A new member of the TEKTIN gene family, which was designated as rat Tektin5, was obtained by PCR technique. Rat Tektin5 cDNA consists of 1,674 bp encoding a 62.8 kDa protein of 558 amino acids. Tektin5 protein contains a Tektin domain as well as a nonapeptide signature sequence that is a prominent feature of Tektin proteins. RT-PCR analysis indicated that Tektin5 was predominantly expressed in testis and that its expression was up-regulated during testis development. Immunoblot analyses revealed that Tektin5 is present in sperm flagella but not in heads and that it is completely released from rat spermatozoa by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Confocal laser scanning microscopy revealed that Tektin5 was located in the middle piece of flagella in rat spermatozoa with no immunolabeling in the heads and the principal piece. Immunogold electron microscopy adopting pre-embedding method discovered that Tektin5 is predominantly associated with the inner side of the mitochondrial sheath. Tektin5 might work as a middle piece component requisite for flagellar stability and sperm motility.     

Channelrhodopsin-1 initiates phototaxis and photophobic responses in chlamydomonas by immediate light-induced depolarization

Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H+-channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H+, CHR1 also conducts Na+, K+, and Ca2+. The kinetic selectivity analysis demonstrates that H+ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1505 and CHR1463, that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.     

Rapid DNA chemical ligation for amplification of RNA and DNA signal

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.     

Fission yeast Swi5 protein, a novel DNA recombination mediator

The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5-Sfr1 and Swi5-Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5-Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5-Sfr1 works independently of another mediator complex, Rhp55-Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.     

Psychological and weight-related characteristics of patients with anorexia nervosa-restricting type who later develop bulimia nervosa

Background  

Over the last five to ten years there has been an increase in psychosomatic complaints (PSC) in Swedish children. The objective of the study was to examine the relation between PSC and sense of coherence (SOC).     

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background

Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.

Methodology/Principal Findings

Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.

Conclusions and Significance

The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.     

Effects of a novel Y5 antagonist in obese mice: combination with food restriction or sibutramine

Objective: To further address the function of the Y5 receptor in energy homeostasis, we investigated the effects of a novel spironolactone Y5 antagonist in diet-induced obese (DIO) mice. Methods and Procedures: Male C57BL/6 or Npy5r^−/− mice were adapted to high-fat (HF) diet for 6–10 months and were submitted to three experimental treatments. First, the Y5 antagonist at a dose of 10 or 30 mg/kg was administered for 1 month to DIO C57BL/6 or Npy5r^−/− mice. Second, the Y5 antagonist at 30 mg/kg was administered for 1.5 months to DIO C57BL/6 mice, and insulin sensitivity was evaluated using an insulin tolerance test. After a recovery period, nuclear magnetic resonance measurement was performed to evaluate body composition. Third, DIO mice were treated with the Y5 antagonist alone, or in combination with 10% food restriction, or with another anorectic agent, sibutramine at 10 mg/kg, for 1.5 months. Plasma glucose, insulin, and leptin levels, and adipose tissue weights were quantified. Results: The spironolactone Y5 antagonist significantly reduced body weight in C57BL DIO mice, but not in Npy5r^−/− DIO mice. The Y5 antagonist produced a fat-selective loss of body weight, and ameliorated obesity-associated insulin resistance in DIO mice. In addition, the Y5 antagonist combined with either food restriction or sibutramine tended to produce greater body weight loss, as compared with single treatment. Discussion: These findings demonstrate that the Y5 receptor is an important mediator of energy homeostasis in rodents.