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Background: The C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs. Results: Due to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC), sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease. Conclusions: These preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind CA-CTD. [ABSTRACT FROM AUTHOR]

Java is an object-oriented programming language. From a software engineering perspective, object-oriented design and programming is used at the architectural design, and structured design and programming is used at the detailed design within methods. As such, structured programming skills are fundamental to more advanced object-oriented programming concepts. Structured programming uses control statements to control the order of execution of a program. In Java, only three forms of control structures are used to implement an algorithm: sequence, selection, and repetition. Sequence structures are a built-in feature: statements run in the order they are listed in the program. Selection structures include single-selections, double-selections, and multiple selections. Repetition structures are implemented in three ways: while statement, for statement, and do--while statement. These simple control structures can be combined into more complex algorithms in only two ways -- stacking and nesting. The purpose of the "Blue, Berry, and Blueberry" assignment is to assess students' structured programming skills with Java. This assignment asks students to write a program that uses all three repetition structures to print, on each line, a number (1 to 100) and a word ("Blue" or "Berry" or "Blueberry", depending on whether the number is divisible by 3 or 5 or 15). The program is also required to report the total numbers of "Blue", "Berry", and "Blueberry". The assignment was given to undergraduate students enrolled in an advanced object-oriented programming course using Java. The submissions for the assignment were graded using a five-criterion rubric. All in all, the class average score of the assignment was about 4.3 out of 5 (86.0%). The top two issues included formatting the output and counting the number of words. To evaluate the students' perceptions of the assignment, an online survey was created, including two essay questions. The first question is: "Do you like the 'Blue, Berry, and Blueberry' assignment? Why or why not?" The second question is: "What are the most difficult challenges that you encountered while working on this assignment?" Each question received 23 responses. In response to question one, more than 90% (21 out of 23) of the respondents said that they did like the assignment, mostly because it helped them to understand the concepts and provided an opportunity to use and compare different forms of selection structures (i.e., if, if--else, and if--else if--else selections) and repetition structures (i.e., while, for, and do--while loops). In response to question two, most of the 23 students felt that the assignment was not all that difficult once they figured out what was expected. The top three most reported challenges include formatting the output, getting the total count for each word, and checking numbers divisible by 15 first. In summary, results from the instructor's grading report and student perceptions indicate that this assignment is effective for assessing students' structured programming skills with Java. [ABSTRACT FROM PUBLISHER]

The toxicity of seven phthalate esters (PAEs), dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butylbenzyl phthalate (BBP), di-n-hexyl phthalate (DnHP), di-(2-ethylhexyl) phthalate (DEHP) and di-n-octyl phthalate (DOP) to embryogenesis and larval development of the marine univalve Haliotis diversicolor supertexta was examined by means of twostage embryo toxicity test. At the blastula stage, the normal embryonic development of H. diversicolor supertexta showed a good dose-response decrease when exposed to DMP, DEP, DBP, BBP, and DnHP. 9-h EC50 values of DMP, DEP, DBP, BBP, and DnHP were 55.71, 39.13, 8.37, 2.65, and 3.32 mg/l, respectively. 9-h EC50 values of DEHP and DOP were not available due to their low solubility. The toxicity order of seven tested PAEs was BBP[DnHP[DBP[DEP[DMP[DOP[DEHP. With the completion of metamorphosis as an experimental endpoint, the 96-h no-observed effect concentration values of DBP, DEHP and the other five tested PAEs were 0.022, 0.021, and 0.020 mg/l, respectively. Due to simple obtainment, convenient stimulation to spawn in the lab, greater sensitivity than mature species, and short culture time, the embryos of H. diversicolor supertexta have the potential to be utilized in acute toxicity test for at least PAEs. solubility. The toxicity order of seven tested PAEs was BBP[DnHP[DBP[DEP[DMP[DOP[DEHP. With the completion of metamorphosis as an experimental endpoint, the 96-h no-observed effect concentration values of DBP, DEHP and the other five tested PAEs were 0.022, 0.021, and 0.020 mg/l, respectively. Due to simple obtainment, convenient stimulation to spawn in the lab, greater sensitivity than mature species, and short culture time, the embryos of H. diversicolor supertexta have the potential to be utilized in acute toxicity test for at least PAEs. [ABSTRACT FROM AUTHOR]

Protein 4.1R (4.1R) is a multifunctional component of the red cell membrane. It forms a ternary complex with actin and spectrin, which defines the nodal junctions of the membrane-skeletal network, and its attachment to the transmembrane protein glycophorin C creates a bridge between the protein network and the membrane bilayer. We now show that deletion of 4.1R in mouse red cells leads to a large diminution of actin accompanied by extensive loss of cytoskeletal lattice structure, with formation of bare areas of membrane. Whereas band 3, the preponderant transmembrane constituent, and proteins known to be associated with it are present in normal or increased amounts, glycophorin C is missing and XK, Duffy, and Rh are much reduced in the 4.1R-deficient cells. The inference that these are associated with 4.1R was borne out by the results of in vitro pull- down assays. Furthermore, whereas Western blot analysis showed normal levels of band 3 and Kell, flow cytometric analysis using an antibody against the extracellular region of band 3 or Kell revealed reduction of these two proteins, suggesting a conformational change of band 3 and Kell epitopes. Taken together, we suggest that 4.1R organizes a macromolecular complex of skeletal and transmembrane proteins at the junctional node and that perturbation of this macro-molecular complex not only is responsible for the well characterized membrane instability but may also remodel the red cell surface. [ABSTRACT FROM AUTHOR]

The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (αII and βII) with those of erythrocyte spectrin (αI and βI). The unfolding transition midpoints (Tm) of the 36 αII- and βII-spectrin repeats extend between 24 and 82 °C, with an average higher by some 10 °C than that of the αI- and βI-spectrin repeats. This difference is reflected in the Tm values of the intact brain and erythrocyte spectrins. Two of three tandem-repeat constructs from brain spectrin exhibited strong cooperative coupling, with elevation of the Tm of the less stable partner corresponding to coupling free energies of approximately -4.4 and -3.5 kcal/mol. The third tandem-repeat construct, by contrast, exhibited negligible cooperativity. Tandem-repeat mutants, in which a part of the "linker" helix that connects the two domains was replaced with a corresponding helical segment from erythroid spectrin, showed only minor perturbation of the thermal melting profiles, without breakdown of cooperativity. Thus, the linker regions, which tolerate few point mutations without loss of cooperative function, have evidently evolved to permit conformational coupling in specified regions. The greater structural stability of the repeats in αII- and βII-spectrin may account, at least in part, for the higher rigidity of brain compared to erythrocyte spectrin. [ABSTRACT FROM AUTHOR]

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Titon X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types. [ABSTRACT FROM AUTHOR]

The article reports on the Fourth International Conference on Watershed Management held in Shenzhen, China in December 2004. The main subjects discussed were urban-watershed management strategies, water treatment technologies and methods to achieve sustainable use of urban water resources. The congress was organized by the Research Center for Environmental Engineering and Management and other institutions.

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (Tm) ranging from 21 to 72 °C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol-1. Constructs comprising five successive repeats from the E-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation. [ABSTRACT FROM AUTHOR]

Microtubules are cytoskeletal polymers consisting of tubulin subunits that take part in diverse cell activities. Many viruses hijack cellular motor proteins to move on microtubules toward the cell interior during the entry process and toward the plasma membrane during the egress period. In addition, viruses often remodel microtubules to facilitate the generation of infectious progeny. In this study, we found that the transactivator of transcription protein of Jembrana disease virus (Jtat) bound tubulin and microtubules both in cells and in the purified system. Microtubule co-sedimentation and co-localization assays revealed a robust interaction of Jtat with microtubules. Tubulin turbidity assay further showed that Jtat promoted tubulin polymerization in vitro in a concentration-dependent manner. Moreover, Jtat promoted the partitioning of cellular tubulin toward the polymeric form, increased the level of tubulin acetylation, and significantly enhanced the cold stability of cellular microtubules. In addition, Jtat-mediated disruption of microtubule dynamics induced the release of Bim from microtubules, leading to profound apoptosis. These results not only identify Jtat as an important viral regulator of microtubule dynamics but also indicate that Jtat-induced apoptosis might contribute to Jembrana disease pathogenesis. [ABSTRACT FROM AUTHOR]