DNA frayed wires: Differential polymerization of d(AnGm) oligonucleotides

Oligodeoxyribonucleotides with terminal runs of contiguous guanines, d(A_nG_m), spontaneously associate into high molecular weight complexes that resolve on polyacrylamide gels as a regular ladder pattern of bands with low mobility. The aggregates, which we call frayed wires, arise from the interaction between the guanine residues of the oligonucleotides; the adenine tracts are single stranded and can take part in Watson–Crick interactions. Oligonucleotides, with different arm‐to‐stem ratios and total length, readily associate in the presence of Mg²⁺ to form aggregates consisting of an integer number of strands. The type of the observed aggregates is determined by the length of the guanine run. Oligonucleotides with six guanines form four‐ and eight‐stranded complexes; there is no further polymerization. An increase in the number of guanine residues to 10 and 15 leads to polymerization resulting in a ladder pattern of up to 9 bands and an intense signal at the top of the gel. The relative population of any given species in a frayed wire sample is governed by the guanine stem length and is not affected to any substantial extent by arms up to 40 bases long. The type and concentration of the cation in the solution affect the degree of aggregation, with Na⁺ and K⁺ promoting the formation of complexes comprised of 2–4 strands and Mg²⁺ being the most effective in facilitating polymerization. The electrophoretic behavior of frayed wires was analyzed in the framework of the Ogston theory. The free mobility of frayed wires in the solution is close to the values reported for single‐stranded DNA, indicating the equivalence of the charge density of the two conformations. The retardation coefficients for frayed wires arising from a single kind of parent strand increase with the introduction of each additional strand. There is no correlation between the retardation coefficient and the type of parent strand; rather, the magnitude of the retardation coefficient is determined by the total molecular weight of the complex. The values of the retardation coefficients are consistently higher than those for double‐stranded DNA and they display much stronger dependence on the total molecular weight. Presumably, the distinct structural and dynamic characteristics of the two conformations account for their different electrophoretic behavior. © 1999 John Wiley & Sons, Inc. Biopoly 49: 287–295, 1999     

An enzyme in the test tube,and a transcription factor in the cell: Moonlighting proteins and cellular factors that affect their behavior

In the cell, expression levels, allosteric modulators, post‐translational modifications, sequestration, and other factors can affect the level of protein function. For moonlighting proteins, cellular factors like these can also affect the kind of protein function. This minireview discusses examples of moonlighting proteins that illustrate how a single protein can have different functions in different cell types, in different intracellular locations, or under varying cellular conditions. This variability in the kind of protein activity, added to the variability in the amount of protein activity, contributes to the difficulty in predicting the behavior of proteins in the cell.     

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL‐induced osteoclastogenesis

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL‐induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F‐actin ring formation and tartrate‐resistant acid phosphatase (TRAP) staining in dose‐dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK‐related trigger RANKL by phosphorylation JNK/ERK/p38‐MAPK, IκBα/p65‐NF‐κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K‐AKT‐NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL‐induced RANK‐TRAF6 association and RANKL‐related gene and protein markers such as NFATc1, Cathepsin K, MMP‐9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.     

Molecular discrimination of structurally equivalent Lys 63‐linked and linear polyubiquitin chains

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63‐linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48‐linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin‐binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF‐κB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.     

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

Opioid receptor-like 1 (ORL1) receptor binding and the biological properties of Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH2 and its analogs.

Hexapeptides such as Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) and Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH(2) have been isolated from a combinatorial peptide library as small peptide ligands for the opioid peptide-like 1 (ORL1) receptor. To investigate the detailed structural requirements of hexapeptides, 25 analogs of these hexapeptides, based on the novel analog Ac-Arg-Tyr-Tyr-Arg-Ile-Arg-NH(2) (1), were synthesized and tested for their ORL1 receptor affinity and agonist/antagonist activity on mouse vas deferens (MVD) tissues. Analog 1 and its Cit(6)-analog (10) were found to possess high affinity to the ORL1 receptor, comparable to that of nociceptin/orphanin FQ, and exhibited potent antagonist activity (pA(2) values of 7.77 for 1 and 7.51 for 10, which are higher than that of [NPhe(1)]nociceptin(1-13)-NH(2) (6.90) on MVD assay. It was also found that the amino acid residue in position 5 plays a key role in agonist/antagonist activity, i.e. an L-configuration aliphatic amino acid is required for potent antagonist activity, while a nonchiral or D-configuration residue produces potent agonist activity. These lines of evidence may provide insight into the mechanisms controlling agonist/antagonist switching in the ORL1 receptor, and may also serve to help developing more potent ORL1 agonists and antagonists.     

EFFECTS OF LIGHT ON PHOTOSYNTHESIS,GRAZING, AND POPULATION DYNAMICS OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

In studying how environmental factors control the population dynamics of Pfiesteria piscicida Steidinger et Burkholder, we examined the influence of light regime on kleptoplastidic photosynthesis, growth, and grazing. Prey (Rhodomonas sp.)‐saturated growth rate of P. piscicida increased (0.67 ± 0.03 d^?1 to 0.91 ± 0.11 d^?1) with light intensity varying from 0 to 200 μmol photons·m^?2·s^?1. No significant effect was observed on grazing, excluding the possibility that light enhanced P. piscicida growth through stimulating grazing. Light‐grown P. piscicida exhibited a higher gross growth efficiency (0.78 ± 0.10) than P. piscicida incubated in the dark (0.32 ± 0.16), and photosynthetic inhibitors significantly decreased growth of recently fed populations. These results demonstrate a role of kleptoplastidic photosynthesis in enhancing growth in P. piscicida. However, when the prey alga R. sp. was depleted, light's stimulating effect on P. piscicida growth diminished quickly, coinciding with rapid disappearance of Rhodomonas‐derived pigments and RUBISCO from P. piscicida cells. Furthermore, the effect of light on growth was reversed after extended starvation, and starved light‐grown P. piscicida declined at a rate significantly greater than dark‐incubated cultures. The observed difference in rates of decline appeared to be attributable to light‐dependent cannibalism. Using a 5‐chloromethylfluorescein diacetate staining technique, cannibalistic grazing was observed after 7 days of starvation, at a rate four times greater under illumination than in the dark. The results from this study suggest that kleptoplastidy enhances growth of P. piscicida only in the presence of algal prey. When prey is absent, P. piscicida populations may become vulnerable to light‐stimulated cannibalism.     

Integration of diffraction phase microscopy and Raman imaging for label‐free morpho‐molecular assessment of live cells

Label‐free quantitative imaging is highly desirable for studying live cells by extracting pathophysiological information without perturbing cell functions. Here, we demonstrate a novel label‐free multimodal optical imaging system with the capability of providing comprehensive morphological and molecular attributes of live cells. Our morpho‐molecular microscopy (3M) system draws on the combined strength of quantitative phase microscopy (QPM) and Raman microscopy to probe the morphological features and molecular fingerprinting characteristics of each cell under observation. While the commonr‐path geometry of our QPM system allows for highly sensitive phase measurement, the Raman microscopy is equipped with dual excitation wavelengths and utilizes the same detection and dispersion system, making it a distinctive multi‐wavelength system with a small footprint. We demonstrate the applicability of the 3M system by investigating nucleated and nonnucleated cells. This integrated label‐free platform has a promising potential in preclinical research, as well as in clinical diagnosis in the near future.