HIV-1 Protease Uses Bi-Specific S2/S2′ Subsites to Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PRs) have a unique specificity that allows cleavage of sites with or without a P1′ proline. A P1′ proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting that PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 combinations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2–P1/P1′–P2′), with two complementary sets of rules. When P1′ is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2′ amino acid interacts with a hydrophobic region of the S2′ subsite. When P1′ is not proline, the orientations of the P2 and P2′ side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite, while the P2′ amino acid usually engages hydrophilic amino acids in the S2′ subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2′ subsites to accommodate the steric effects imposed by a P1′ proline on the orientation of P2 and P2′ substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.     

限气贮藏猕猴桃果实伤害产生机制的研究

采用ＰＥ袋方法于常温一藏秦猕猴桃,贮藏期间袋内ＣＯ２浓度高于６％时果实会产生伤害贮藏前期,果实的果肉和果心硬度均很高,这与两部位淀粉酶和多聚半乳糖醛酸酶活性严重被抑制淀粉和果胶物质水解被抑制有关。贮藏中期,果肉出现水浸状,到贮藏末期,果心和照相机人均很快腐烂,这主要与Ｖｃ含量明显降低,导致膜脂过氧化加剧有关。而与ＳＯＤ活性变化关系不大。而贮藏期间,果心硬度明显高于果肉,这与果心淀粉酶和多聚半乳醛酸     

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating T_m for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/ . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The S1 split signal of photosystem II; a tyrosine-manganese coupled interaction

Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q_A⁻ is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (< 5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680⁺ is observed with the same decay kinetics as the S₁ split signal. Cryogenic donors to P680⁺ seen are: (i) transient (t_1/2 ∼ 150 s) tyrosine related species, including ‘split signals’ (∼ 15% total centers), (ii) reduced cytochrome b₅₅₉ (∼ 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, (∼ 30% centers).     

Herbal monoterpene alcohols inhibit propofol metabolism and prolong anesthesia time

2,6-Diisopropylphenol (Propofol) is a short-acting intravenous anesthetic that is rapidly metabolized by glucuronidation and ring hydroxylation catalyzed by cytochrome P450. The goal of this research was to determine whether dietary monoterpene alcohols (MAs) could be used to prolong the anesthetic effect of propofol by inhibiting propofol metabolism in animals. Mice were injected intraperitoneally (i.p.) with MAs (100-200) mg/kg followed by the administration of 100 mg/kg propofol 40 min later via an i.p. injection. The time of the anesthesia of each mouse was recorded. It was found that (+/-)-borneol, (-)-carveol, trans-sobrerol, and menthol significantly extended the anesthetic effect of propofol (>3 times). The concentration of propofol in the mouse blood over time (up to 180 min) also increased in mice pre-treated with (-)-borneol, (-)-carveol, and trans-sobrerol. The volume of distribution of propofol decreased in the (-)-borneol (p<0.05), pre-treated group as compared to the propofol control group. Moreover, the maximum blood concentration of propofol and the concentration of propofol in the blood as indicated by the area under the curve were significantly increased in (-)-borneol and (-)-carveol pre-treated groups. Additional evidence using rat hepatocytes showed that (-)-borneol inhibited propofol glucuronidation whereas trans-sobrerol and (-)-carveol inhibited cytochrome P450 dependent microsomal aminopyrine N-demethylation. These results suggest that (-)-borneol extends propofol-induced anesthesia by inhibiting its glucuronidation in the mouse whereas trans-sobrerol (-)-carveol extends propofol-induced anesthesia by inhibiting P450 catalyzed propofol metabolism.     

蓝舌病毒HbC3株对小鼠乳腺癌MA-782细胞的感染特性

体外研究蓝舌病毒湖北株3(BTV-HbC3)对鼠乳腺癌细胞MA782的感染性并探讨BTV-HbC3靶向性溶癌的机制。观察MA782细胞感染BTV-HbC3的病变效应(CPE);MTT法研究病毒致细胞病变率的特征;透射电镜观察感染病毒后细胞超微结构的变化;免疫组化研究病毒蛋白在细胞内的表达特点;凋亡染色(TUNEL法)观察病毒诱导细胞凋亡的情况;流式细胞仪测定病毒对MA782细胞周期的影响。结果证明BTV-HbC3感染MA782细胞后有明显的细胞病变效应;病毒蛋白主要表达在细胞的胞膜及胞浆内;凋亡染色发现大量的凋亡细胞;流式细胞仪可见明显的亚二倍体峰。故认为BTV-HbC3在体外能有效的感染MA782细胞,并能诱导MA782细胞凋亡。     

Parent-offspring conflict and the evolution of infant-directed song

We present a theory of the origin and evolution of infant-directed song, a form of music found in many cultures. After examining the ancestral ecology of parent-infant relations, we propose that infant-directed song arose in an evolutionary arms race between parents and infants, stemming from the dynamics of parent-offspring conflict. We describe testable predictions that follow from this theory, consider some existing evidence for them, and entertain the possibility that infant-directed song could form the basis for the development of other, more complex forms of music.     

HIV-1 Envelope Glycoprotein Biosynthesis, Trafficking, and Incorporation

The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor (gp160) that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly, the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and coreceptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation and the role of specific membrane microdomains in this process. Here, we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions.     

Visualizing HIV-1 Assembly

The assembly of an HIV-1 particle is a complex, multistep process involving several viral and cellular proteins, RNAs and lipids. While many macroscopic and fixed-cell microscopic techniques have provided important insights into the structure of HIV-1 particles and the mechanisms by which they assemble, analysis of individual particles and their assembly in living cells offers the potential of surmounting many of the limitations inherent in other approaches. In this review, we discuss how the recent application of live-cell microscopic imaging techniques has increased our understanding of the process of HIV-1 particle assembly. In particular, we focus on recent studies that have employed total internal reflection fluorescence microscopy and other single-virion imaging techniques in live cells. These approaches have illuminated the dynamics of Gag protein assembly, viral RNA packaging and ESCRT (endosomal sorting complex required for transport) protein recruitment at the level of individual viral particles. Overall, the particular advantages of individual particle imaging in living cells have yielded findings that would have been difficult or impossible to obtain using macroscopic or fixed-cell microscopic techniques.