用二次电泳法研究核酸与蛋白质的相互作用

研究蛋白质与核酸的结合常遇到的问题是对蛋白质等电点及可溶度等要求较高,或难以同时处理大量标本。为克服此缺点,将待检蛋白经聚丙烯酰胺凝胶电泳后,通过洗涤去除凝胶中的ＳＤＳ,使蛋白质相对固定于凝胶中,改电泳液为ＴＡＥ或ＴＢＥ,继之用同位素标记寡核苷酸进行二次电泳,通过放射自显影直观地显现出蛋白结合核酸的结果。该法敏感,特异,对蛋白质等电点及可溶性要求低,可同时检测多个样本,值得推广使用。     

Response to biostimulation in peri-puberal beef heifers: influence of male-female proximity and heifer's initial body weight

The objectives were to determine if exposure of 12 mo old peri-puberal beef heifers to androgenized steers for 35 d hastened puberty, and if the response was related to the physical proximity between males and females and to heifer's initial body weight. Hereford x Aberdeen Angus heifers (n = 131), 12 mo old, were assigned to two treatments: 1) Exposed group, exposed to androgenized steers from Day 0 to Day 35 (E, n = 66); or 2) Control group, isolated from the steers and other males (C, n = 65). Cyclic activity was determined through estrous behavior detection (twice daily) and weekly ultrasound imaging to detect a CL. For each Exposed heifer, an association index (with males) was determined thrice weekly, based on the distance to androgenized steers every 10 min for 4 h (until cyclic activity began). Data were analyzed according to heifer's initial body weight, which was categorized into three ranges (low, medium, and high, designated LW, MW, and HW, respectively). The cumulative proportion of cyclic heifers was greater for Exposed than Control heifers as of Day 21. By the end of the exposure period, more Exposed than Control heifers had attained puberty (16/66 vs. 2/65; P < 0.001). Within the HW classification, more Exposed than Control heifers became puberal (11/20 vs. 2/21; P = 0.002). However, there were no differences between MW and LW in the proportions of heifers that reached puberty. Association index in Exposed heifers was greater in HW than in MW and LW (0.10 ± 0.09 vs. 0.06 ± 0.03 and 0.06 ± 0.04; P < 0.05), and in heifers that began cyclic activity compared to those that did not in the MW heifers (0.09 ± 0.05 vs. 0.05 ± 0.02; P = 0.01) and tended to be different in the HW treatment (0.12 ± 0.10 vs. 0.06 ± 0.02; P = 0.09). In conclusion, exposure of peri-puberal beef heifers to androgenized steers for 35 d advanced puberty in heavier heifers; an earlier response occurred in heifers with greater proximity to androgenized steers.     

A Comparative Study of Fibroblast Behaviors under Cyclic Stress Stimulus and Static Culture on 3D Patterned Matrix

Mechanical stress and patterned surface of the scaffolds has been recognized as a crucial factor in determining cell functionality and tissue development, which in turn can direct the cell responses. In this study, fibroblasts M-3T3 in three-dimensional (3D) honeycomb patterning Chitosan/Poly(L-Lactic Acid) (CS/PLLA) composites was stimulated by a 15% sinusoidal (1 Hz) strain applied by a biodynamic test instrument. The effects of mechanical stimulus on the cell proliferation and basic Fibroblast Growth Factor (bFGF) secretion were studied in comparison to the non-strain groups and blank control. Results show that fibroblasts are able to sense the mechanical stimulation and respond, resulting in a time dependent increase of bFGF secretion and promoting cell proliferation. Moreover, the cells seeded in the scaffolds showed a higher cell proliferation and bFGF secretion. These findings support the hypothesis that suitable mechanical stimulus has positive effect on fibroblasts, and such a 3D honeycomb patterned scaffold may play a positive role in regulating cell behaviors in vitro.     

Genome-specific control of meiotic pairing evidenced in mutant Aegilops ventricosa-Secale cereale amphidiploids

Summary The Ae. ventricosa and S. cereale genomes were distinguished at meiosis by the C-banding procedure. Only two plants of the amphiploid Ae. ventricosa-S. cereale were found to exhibit the high degree of asynapsis limited to Aegilops ventricosa genomes. In addition, these genomes showed higher homoeologous pairing than homologous pairing frequencies. These results can be explained by the existence of separate genome-specific control of meiotic pairing between the chromosomes of both species in these synaptic mutant plants.     

Simultaneous Non‐inferiority Test of Sensitivity and Specificity for Two Diagnostic Procedures in the Presence of a Gold Standard

Sensitivity and specificity have traditionally been used to assess the performance of a diagnostic procedure. Diagnostic procedures with both high sensitivity and high specificity are desirable, but these procedures are frequently too expensive, hazardous, and/or difficult to operate. A less sophisticated procedure may be preferred, if the loss of the sensitivity or specificity is determined to be clinically acceptable. This paper addresses the problem of simultaneous testing of sensitivity and specificity for an alternative test procedure with a reference test procedure when a gold standard is present. The hypothesis is formulated as a compound hypothesis of two non‐inferiority (one‐sided equivalence) tests. We present an asymptotic test statistic based on the restricted maximum likelihood estimate in the framework of comparing two correlated proportions under the prospective and retrospective sampling designs. The sample size and power of an asymptotic test statistic are derived. The actual type I error and power are calculated by enumerating the exact probabilities in the rejection region. For applications that require high sensitivity as well as high specificity, a large number of positive subjects and a large number of negative subjects are needed. We also propose a weighted sum statistic as an alternative test by comparing a combined measure of sensitivity and specificity of the two procedures. The sample size determination is independent of the sampling plan for the two tests.     

Gen‐morph species concept—A new and integrative species concept for outbreeding organisms

The present article briefly reviewed the prevailing species concepts, especially biological, genetic, evolutionary, phylogenetic, ecological, and several taxonomic species concepts. The former five reflect the properties of species from diverse aspects and in different degrees, while taxonomic species concepts all contain more or less subjective elements, except for Hedberg's taxonomic method (not species concept). So far, there is no species concept that is both theoretically rational and practically operable. The present article outlined recent studies on the genus Paeonia L. (Paeoniaceae) in biology, particularly in morphology, biogeography, molecular phylogeny, and reproductive behavior, which provided insight into the relationship between variation of morphological characteristics and phylogeny. Taking the study on Paeonia L. as a case, referring to studies on some other plant groups, and incorporating the merits of the prevailing species concepts into our consideration, “gen‐morph species concept” is proposed here formally as new for outbreeding organisms. The new species concept has three special features: (i) a bridge linking morphological aspect with genetic and other aspects of species; (ii) proposal of a concrete morphological criterion for species definition, and (iii) considering quantitative and qualitative characteristics as equally valuable for species definition and introducing statistics into the concept to handle such characteristics. Therefore, the gen‐morph species concept is an integrative species concept, both theoretically objective and practically operable.     

Pancreas and Kidney Transplantation Using Embryonic Donor Organs

One novel solution to the shortage of human organs available for transplantation envisions ‘growing’ new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. We and others have shown that renal anlagen (metanephroi) transplanted into animal hosts undergo differentiation and growth, become vascularized by blood vessels of host origin and exhibit excretory function. Metanephroi can be stored for up to 3 days in vitro prior to transplantation with no impairment in growth or function post-implantation. Metanephroi can be transplanted across both concordant (rat to mouse) and highly disparate (pig to rodent) xenogeneic barriers. Similarly, pancreatic anlagen can be transplanted across concordant and highly disparate barriers, and undergo growth, differentiation and secrete insulin in a physiological manner following intra-peritoneal placement. Implantation of the embryonic pancreas, is followed by selective differentiation of islet components. Here we review studies exploring the potential therapeutic applicability for organogenesis of the kidney or endocrine pancreas.     

Synthesis and General Biological Activity of a Small Adenosine-5′-(Carboxamide and Sulfanilamide) Library

A small library of fifty-five adenosine peptide analogs was synthesized, under the Pilot Scale Library (PSL) Program of the NIH Roadmap initiative, from 2′,3′-O-isopropylideneadenosine-5′-carboxylic acid 2. The coupling of amine or sulfanilamide reactants to the free 5′-carboxylic acid moiety of 2, in automated solution-phase fashion, led after acid-mediated hydrolysis to target compounds 3–57 in good yields and high purity. No marked anticancer or antimalarial activity was noted on preliminary cellular testing. Initial screening through the MLPCN program, however, indicates that these analogs may show diverse and interesting biological activities.     

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.