Anhydrobiosis in tardigrades--the last decade

The current state of knowledge about anhydrobiosis in tardigrades is presented. In response to adverse environmental conditions tardigrades arrest their metabolic activity and after complete dehydration enter the so-called “tun” state. In this ametabolic state they are able to tolerate exposure to various chemical and physical extremes. These micrometazoans have evolved various kinds of morphological, physiological and molecular adaptations to reduce the effects of desiccation. In this review we address behavioral adaptation, morphological features and molecules which determine the anhydrobiotic survival. The influence of the time spent in anhydrobiotic state on the lifespan and DNA and the role of the antioxidant defense system are also considered. Finally we summarize recent input from the “omics” sciences.     

Hybridization Properties of RNA Containing 8-Methoxyguanosine and 8-Benzyloxyguanosine

Modified nucleobase analogues can serve as powerful tools for changing physicochemical and biological properties of DNA or RNA. Guanosine derivatives containing bulky substituents at 8 position are known to adopt syn conformation of N-glycoside bond. On the contrary, in RNA the anti conformation is predominant in Watson-Crick base pairing. In this paper two 8-substituted guanosine derivatives, 8-methoxyguanosine and 8-benzyloxyguanosine, were synthesized and incorporated into oligoribonucleotides to investigate their influence on the thermodynamic stability of RNA duplexes. The methoxy and benzyloxy substituents are electron-donating groups, decreasing the rate of depurination in the monomers, as confirmed by N-glycoside bond stability assessments. Thermodynamic stability studies indicated that substitution of guanosine by 8-methoxy- or 8-benzyloxyguanosine significantly decreased the thermodynamic stability of RNA duplexes. Moreover, the presence of 8-substituted guanosine derivatives decreased mismatch discrimination. Circular dichroism spectra of modified RNA duplexes exhibited patterns typical for A-RNA geometry.     

Crystal structure of RuvC resolvase in complex with Holliday junction substrate

The key intermediate in genetic recombination is the Holliday junction (HJ), a four-way DNA structure. At the end of recombination, HJs are cleaved by specific nucleases called resolvases. In Gram-negative bacteria, this cleavage is performed by RuvC, a dimeric endonuclease that belongs to the retroviral integrase superfamily. Here, we report the first crystal structure of RuvC in complex with a synthetic HJ solved at 3.75 Å resolution. The junction in the complex is in an unfolded 2-fold symmetrical conformation, in which the four arms point toward the vertices of a tetrahedron. The two scissile phosphates are located one nucleotide from the strand exchange point, and RuvC approaches them from the minor groove side. The key protein–DNA contacts observed in the structure were verified using a thiol-based site-specific cross-linking approach. Compared with known complex structures of the phage resolvases endonuclease I and endonuclease VII, the RuvC structure exhibits striking differences in the mode of substrate binding and location of the cleavage site.     

The virulence of Staphylococcus aureus correlates with strain genotype in a chicken embryo model but not a nematode model

Staphylococcus aureus infections are of major importance in human and veterinary medicine. Studies of the virulence of this bacterium are complicated by inconsistent results obtained in different animal models. We searched for an uncomplicated and inexpensive model suitable to study virulence of poultry strains of S. aureus using a genome-wide approach. We determined that a useful model would clearly differentiate strains of high and low virulence, and that this would generally correlate with the genetic relatedness among strains. To this end Gallus gallus (chicken) embryo and Caenorhabditis elegans (nematode) models were selected, and their response to challenge by a set of well-characterized Staphylococcus strains was evaluated. The chicken embryo model allowed to determine variation in virulence among strains of poultry and human origin. The survival of embryos ranged from 0% to almost 100% for the various strains. In contrast, variation in virulence of most strains in the nematode model was comparable, regardless of their origin or genotype, demonstrating limited usefulness of this model. Most importantly, a clear correlation was found between the virulence level in the embryo model and the genotype of the tested poultry strains. Our findings indicate the potential usefulness of embryo model for future identification of host-specific adaptations and virulence factors in S. aureus.     

Light and scanning microscopic studies of integument differentiation in the grass snake Natrix natrix L. (Lepidosauria,Serpentes) during embryogenesis

We analysed the differentiation of body cover in the grass snake (Natrix natrix L.) over the full length of the embryo's body at each developmental stage. Based on investigations using both light and scanning electron microscopes, we divided the embryonic development of the grass snake integument into four phases. The shape of the epidermal cells changes first on the caudal and ventral parts of the embryo, then gradually towards the rostral and dorsal areas. In stage V on the ventral side of the embryo the gastrosteges are formed from single primordia, but on the dorsal side the epidermis forms the scale primordia in stage VII. This indicates that scalation begins on the ventral body surface, and spreads dorsally. The appearance of melanocytes between the cells of the stratum germinativum in stage VII coincides with changes in embryo colouration. The first dermal melanocytes were detected in stage XI so in this stage the definitive skin pattern is formed. In the same stage the epidermis forms the first embryonic shedding complex and the periderm layer begins to detach in small, individual flakes. This process coincides with rapid growth of the embryos.     

Metabolism of cyclic ADP-ribose: Zinc is an endogenous modulator of the cyclase/NAD glycohydrolase ratio of a CD38-like enzyme from human seminal fluid

CD38, a bifunctional enzyme capable of both synthesis and hydrolysis of the second messenger cyclic ADP-ribose (cADPR). Using the natural substrate of the enzyme, NAD+, the ratio of ADP-ribosyl cyclase/NAD glycohydrolase of CD38 is about 1/100. Here we describe that human seminal fluid contain a soluble CD38 like enzyme with an apparent M.W. of 49 kDa. When purified this enzyme has a cyclase/NAD glycohydrolase ratio of about 1/120. However, the in situ cyclase/NAD glycohydrolase ratio measured in seminal plasma approaches 1/1. We also found that physiological concentrations of zinc present in the seminal fluid, in the range of 0.6 to 4 mM, are responsible for the modulation of the cyclase/NAD glycohydrolase ratio. This new information indicates that the cyclase/NAD glycohydrolase ratio can be modified in vivo.     

Thermodynamic Features of Structural Motifs Formed by β-L-RNA

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in β-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2′-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by ¹H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.