Interfering Residues Narrow the Spectrum of MLV Restriction by Human TRIM5α

TRIM5α is a restriction factor that limits infection of human cells by so-called N- but not B- or NB-tropic strains of murine leukemia virus (MLV). Here, we performed a mutation-based functional analysis of TRIM5α-mediated MLV restriction. Our results reveal that changes at tyrosine³³⁶ of human TRIM5α, within the variable region 1 of its C-terminal PRYSPRY domain, can expand its activity to B-MLV and to the NB-tropic Moloney MLV. Conversely, we demonstrate that the escape of MLV from restriction by wild-type or mutant forms of huTRIM5α can be achieved through interdependent changes at positions 82, 109, 110, and 117 of the viral capsid. Together, our results support a model in which TRIM5α-mediated retroviral restriction results from the direct binding of the antiviral PRYSPRY domain to the viral capsid, and can be prevented by interferences exerted by critical residues on either one of these two partners.     

A lead-dependent DNAzyme with a two-step mechanism

A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg(2+) (called 8-17), 0.5 mM Mg(2+)/50 mM histidine (called Mg5), or 100 microM Zn(2+) (called 17E), all variants are shown to be the most active with Pb(2+) (8-17: k(obs) approximately 0.5 min(-1); Mg5: k(obs) approximately 2 min(-1); 17E: k(obs) approximately 1 min(-1) with 200 microM Pb(2+) at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb(2+) > Zn(2+) > Mn(2+) approximately Co(2+) > Ni(2+) > Mg(2+) approximately Ca(2+) > Sr(2+) approximately Ba(2+). The catalytic rate is half-maximal at 13.5 microM Pb(2+), 0.97 mM Zn(2+), or 10.5 mM Mg(2+), suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb(2+) > Zn(2+) > Mg(2+). The Pb(2+)-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is approximately 1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G*T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg(2+) and Zn(2+) catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb(2+) catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb(2+)-binding sites and their roles in the two-step mechanism.     

Strip test for bedside detection of interleukin-6 in cervical secretions is predictive for impending preterm delivery

Inflammatory cytokines in amniotic fluid are markers of prematurity which could characterize preterm labour of infectious origin. To avoid amniocentesis, we analyzed IL-6, IL-8, IL-10, and IL-13 by RT-PCR in cervical secretions (CS) of 307 women with preterm labour. IL-6 was detected in 26.3% patients who delivered at less than 34 weeks (specificity: 95.8%). In addition, IL-6 was associated with delivery within 7 days (specificity: 91.6%). To render the detection more rapid and cheaper, a strip test was designed and evaluated comparatively with RT-PCR in 76 women. This bedside strip test was twice more sensitive than RT-PCR, with little decrease in specificity.     

Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.     

A note: ortho-phthalaldehyde: proposed mechanism of action of a new antimicrobial agent

Ortho-phthalaldehyde (OPA) is a new aromatic dialdehyde antimicrobial agent, the mechanism of action of which has been little studied. The aims of this paper are to examine what is currently known about its mechanism of action, to compare the action with that of a widely investigated aliphatic dialdehyde, glutaraldehyde (GTA), and to put forward a hypothesis that would, in the light of current knowledge, explain how OPA inactivates micro-organisms, including GTA-resistant Mycobacterium chelonae.     

Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ～200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.     

For she that hath, to her shall be given…Implications of flowering in Anemone nemorosa L

We looked for life‐history trade‐offs between flowering, vegetative growth and somatic maintenance in the common woodland herb Anemone nemorosa. A. nemorosa forms a horizontal rhizome system consisting of previously formed annual segments and terminated by a flowering or non‐flowering shoot. Resources acquired by the aboveground parts are used for flowering, seed production, storage and growth of the annual segments. Resources stored in the rhizome during the growing period are used for preformation of buds, somatic maintenance between two growing periods and development of aboveground parts in the following spring. We hypothesised that the decision to invest in flower buds depends on the amount of resources stored in the recently formed annual segment. We also hypothesised a trade‐off between flowering and segment growth and, finally, as a consequence, we expected individual rhizomes to alternate between the flowering and the non‐flowering state. We found that segments producing flower buds were significantly longer than non‐flowering segments, indicating that resource level influences the function of the preformed buds. Contrary to our expectations, we found flowering rhizomes produced longer annual segments than non‐flowering rhizomes. We suggest the larger leaf area of flowering rhizomes and occasional abortion of flowers or seeds as possible mechanisms behind this pattern. Our study shows that even though the decision to produce a flower bud is taken in another time‐frame than that in which the actual flowering and fruiting takes place, an ostensibly inexpedient decision is changed to a neutral or even an advantageous incident.