Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators

In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)–mediated homologous recombination (HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1–mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA     

Backbone and side-chain 1H, 15N and 13C assignments of mouse peptide ESP4

A peptide or a small protein released from an exocrine gland or in urine is utilized as a chemosignal that elicits social or reproductive behavior in mice. Recently, we identified the male-specific peptide, exocrine gland-secreting peptide 1 (ESP1), in mouse tear fluids that enhanced female sexual receptive behavior, and determined the three dimensional structure. ESP1 appears to be a member of multigene family that consists of 38 genes in mice, which we call the ESP family. ESP4, a member of the ESP family, is expressed in various exocrine glands, and shows the highest sequence similarity with ESP1. Here, we report the NMR assignments of ESP4 which provides a basis for NMR analyses of this protein. Our results will give insight into structural relationships within the ESP family.     

Investigation of Digestion and Absorption of a Dietary Protein by Using Its Antigenicity as an Index: Gastrointestinal Digestion of Ovalbumin

An attempt was made to use the disappearance of ovalbumin antigen as an index for the analysis of the process of digestion of ovalbumin in vitro and in vivo. The antigenic determinants of ovalbumin were highly sensitive to conformational changes in the protein. Mild heat-treatment and acid-denaturation abolished spontaneously all the reactivities of antigenic determinants. Experiments on protease digestion in vitro showed that native ovalbumin was hardly digested by pepsin at pH 2.7 or by pancreatin at pH 8.0, while acid- or heat-denatured ovalbumin was more easily digested by these proteases. The results suggested that the denaturation of ovalbumin was a rate-limiting step in its digestion, and the disappearance of ovalbumin antigen was a useful index of the initial process of digestion which preceded the proteolytic hydrolysis.

At 1 hr after the administration of an ovalbumin diet to rats, approximately 20% of the ovalbumin antigen administered was detected in the whole contents of the gastrointestinal tract. In the small intestine, the amount of intact ovalbumin comprised about half of the total protein of the intestinal contents. These results suggested that in the stomach and small intestine, denaturation of ovalbumin was a significant rate-limiting step in its digestion process. But after 2hr, little ovalbumin antigen was detected in the small intestine. This disappearance of the antigen from the small intestine cannot be well explained by the in vitro data.     

Binding site mapping of a photoaffinity-labeled juvenile hormone binding protein.

The juvenile hormone binding protein (JHBP) of larval Manduca sexta was labeled by a photoaffinity analog of JH II and purified by preparative IEF and ion-exchange HPLC. The purified [3H]EHDA-labeled JHBP was selectively cleaved by CNBr and by endoproteinases Lys-C and Glu-C. The radioactive peptides were separated by tricine SDS-PAGE and sequenced after blotting to a PVDF membrane. The sequence revealed that Ala184-Asn226 contained a primary binding site of [3H]EHDA. Furthermore, peptide mapping indicated that Asp1-Glu34 also contained a second covalent attachment site of [3H]EHDA. Labeling of the N-terminal region increased when the photolysis was performed at lower temperature. Since Ala184-Asn226 is predicted to be a hydrophobic beta-sheet region, it may participate in the recognition of lipophilic backbone of JH. Five out of six cysteines are located in these two regions, consistent with a model in which the two binding regions connected by disulfide bridges provide a two-sided binding pocket for juvenile hormone.     

Tracing Tellurium and Its Nanostructures in Biology

The purpose of this study was to investigate whether the intakes of some kinds of vitamins and minerals are associated with periodontitis in a nationally representative sample of young adults. This study comprised 2049 young adults aged 19–39 years who took both periodontal examination and nutrition survey. The vitamin and mineral intakes were calculated from dietary intake data gained by complete one-day 24-h recall interviews, and the intake levels for each nutrient were classified by the Recommended Nutrient Intake (RNI) in Dietary Reference Intakes for Koreans and median values. Periodontitis was assessed using Community Periodontal Index (CPI). Multivariate logistic regression analyses were performed in a whole sample and subgroups with the strata of gender or smoking, following a complex sampling design. In analyses according to RNI, a lower intake of niacin was significantly associated with periodontitis in young adults (odd ratio [OR] 1.47, 95% confidential interval [CI] 1.09–2.00) and in its subgroup of women (OR 1.70; 95% CI 1.10–2.64) and current non-smokers (OR 1.75; 95% CI 1.22–2.51). Whereas, in analyses according to median intake values, there were significant associations of periodontitis with a lower intake of niacin in women (OR 1.58; 95% CI 1.02–2.46) and current non-smokers (OR 1.50; 95% CI 1.01–2.22), with lower intake of vitamin C in women (OR 1.66; 95% CI 1.04–2.64) and in current non-smokers (OR 1.49; 95% CI 1.04–2.14), with lower intake of iron in women (OR 1.85; 95% CI 1.11–3.07), and with lower intake of vitamin A marginally in women (OR 1.56; 95% CI 1.00–2.44). In young adults, periodonitis is significantly associated with the lower intakes of niacin, vitamin C, and iron, especially in women and current non-smokers.     

Expression of a Gene Specific for Iron Deficiency (Ids3) in the Roots of Hordeum vulgare

To clone genes required for the synthesis of mugineic acid (MA)or for the transport of Fe(III)-MA, a     

Multi-scale modelling of amyloid formation from unfolded proteins using a set of theory derived rate constants

Traditional simulation and analysis of amyloid aggregation kinetics has involved the examination of a single lumped parameter taken to reflect the total mass of protein in amyloid form. However use of increasingly sophisticated multi-experimental strategies capable of providing information on the structure of the growing fibril at the mesoscopic and atomistic level, has put extra information within the experimenter's reach. Although such data can be presented empirically, its incorporation into a theoretical model is more problematic due to scaling issues associated with modern day approaches which fall into either the particle based or statistical based categories. Here we present a coarse grained multi-scale simulation of irreversible amyloid formation that straddles this simulation divide by using a set of theory derived size and conformation specific rate constants to simulate the kinetic evolution of the amyloid fibril population. This approach represents a potentially profitable simulation/analytical strategy that will help to probe more deeply into the underlying molecular driving forces behind the phenomenon of amyloid formation.     

Evaluating the influence of temperature on proanthocyanidin biosynthesis in developing grape berries (Vitis vinifera L.)

Molecular Biology Reports - The variability in grape (Vitis vinifera L.) proanthocyanidin content is largely attributable to viticultural and environmental conditions. However, the particular...     

Immunogenicity of repeated Sendai viral vector vaccination in macaques

Induction of durable cellular immune responses by vaccination is an important strategy for the control of persistent pathogen infection. Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. Repeated vaccination may contribute to durable memory T-cell induction, but anti-vector antibodies could be an obstacle to its efficacy. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient T-cell induction in macaques. Here, we examined whether repeated SeV vector vaccination with short intervals can enhance antigen-specific CD8⁺ T-cell responses. Four rhesus macaques possessing the MHC-I haplotype 90-120-Ia were immunized three times with intervals of three weeks. For the vaccination, we used replication-defective F-deleted SeV vectors inducing CD8⁺ T-cell responses specific for simian immunodeficiency virus Gag_206–216 and Gag_241–249, which are dominant epitopes restricted by 90-120-Ia-derived MHC-I molecules. All four animals showed higher Gag_206–216-specific and Gag_241–249-specific CD8⁺ T-cell responses after the third vaccination than those after the first vaccination, indicating enhancement of antigen-specific CD8⁺ T-cell responses by the second/third SeV vector vaccination even with short intervals. These results suggest that repeated SeV vector vaccination can contribute to induction of efficient and durable T-cell responses.