Differential Intracellular Compartmentalization of Herpetic Thymidine Kinases (TKs) in TK Gene-Transfected Tumor Cells: Molecular Characterization of the Nuclear Localization Signal of Herpes Simplex Virus Type 1 TK

The thymidine kinases (TKs) of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) were expressed in human osteosarcoma cells as fusion proteins with the green fluorescent protein (GFP), and their intracellular localizations were determined. The three TK-GFP fusion products were localized in different subcellular compartments of the transfected tumor cells. HSV-1 TK-GFP was localized exclusively in the nucleus, HSV-2 TK-GFP was predominantly found in the cytosol, while VZV TK-GFP was localized in both the nucleus and the cytosol. In support of these findings, we identified a nuclear localization signal (NLS) in the N-terminal arginine-rich region of HSV-1 TK that was absent in HSV-2 and VZV TK. The first 34 amino acids proved necessary for the specific nuclear localization of HSV-1 TK and, when added to the VZV TK-GFP gene construct, also sufficed to specifically target VZV TK-GFP to the nucleus. Further analysis of this NLS through site-directed mutagenesis revealed that the basic amino acid-rich nonapeptide ²⁵R-R-T-A-L-R-P-R-R³³ is of crucial importance in the nuclear targeting of HSV-1 TK. In particular, we revealed that the presence of the arginine residues at positions 25, 26, 30, 32, and 33 is obligatory for efficient NLS functioning, whereas arginine and histidine residues outside of the nonapeptide (i.e., residues R18, R20, and H22) did not change the functional properties of the NLS.     

Sperm head membrane reorganisation during capacitation

The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Junctional protein ring structures seal the surface of the mid-piece from the head and the tail respectively and probably prevent random diffusion of membrane molecules over the protein rings. Despite the absence of such lateral diffusion-preventing structures, the sperm head surface is also highly heterogeneous. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood. Nevertheless, it is known that each of the sperm head surface regions has specific roles to allow sperm to fertilize the oocyte: a specific region is devoted to zona pellucida binding, a larger area of the sperm head surface is involved in the acrosome reaction (intracellular fusion), while yet another region is involved in egg plasma membrane binding and fertilization fusion (intercellular membrane fusion). All three events occur in the area of the sperm head where the plasma membrane covers the acrosome. Recently, lipid ordered microdomains (lipid rafts) were discovered in membranes of many biological specimens including sperm. In this review, we cover the latest insights about sperm lipid raft research and discuss how sperm lipid raft dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction.     

Liver X Receptor (LXR) activation negatively regulates visfatin expression in macrophages

Adipose tissue macrophages (ATM) are the major source of visfatin, a visceral fat adipokine upregulated during obesity. Also known to play a role in B cell differentiation (pre-B cell colony-enhancing factor (PBEF)) and NAD biosynthesis (nicotinamide phosphoribosyl transferase (NAMPT)), visfatin has been suggested to play a role in inflammation.Liver X Receptor (LXR) and Peroxisome Proliferator-Activated Receptor (PPAR)γ are nuclear receptors expressed in macrophages controlling the inflammatory response. Recently, we reported visfatin as a PPARγ target gene in human macrophages. In this study, we examined whether LXR regulates macrophage visfatin expression. Synthetic LXR ligands decreased visfatin gene expression in a LXR-dependent manner in human and murine macrophages. The decrease of visfatin mRNA was paralleled by a decrease of protein secretion. Consequently, a modest and transient decrease of NAD⁺ concentration was observed. Interestingly, LXR activation decreased the PPARγ-induced visfatin gene and protein secretion in human macrophages.Our results identify visfatin as a gene oppositely regulated by the LXR and PPARγ pathways in human macrophages.     

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target <Emphasis Type="Italic">lox</Emphasis>-Sites

Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were obtained. The efficiency of somatic deletion decreased with increasing deletion size. The largest germinally transmitted deletion had a size of only 55 kb. The results show that Cre-mediated deletion in somatic cells is less efficient when the lox sites are separated over larger distances. A further drop of the deletion efficiency after germinal transmission of the larger deletions can be explained by the probable loss of genes that are of vital importance to gametophyte function. Plasmid rescue of an 8.4 kb circularised deleted DNA showed that the Cre-mediated deletion takes place in tomato as expected. Since the circular Cre-deleted DNA could only be PCR amplified in plant cells where the deletion was not complete, the double-stranded DNA circle is assumed to be instable.     

Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage

The present study describes the effects of exposure of bovine sperm to mild and more intense ROS generating conditions. The membrane integrity of the incubated sperm was assessed and the incubated sperm were used for IVF after which the percentages of cleavage and blastocyst formation were determined for a period up to 9 days. The incubated sperm samples showed increased levels of molecular oxidation in the plasma membrane, the mitochondria, the cytosol and to a lesser extent in the sperm's DNA. The sperm membrane integrity as well as the first cleavage rates obtained with sperm from mild ROS generating conditions (100 microM H2O2) were not different from sperm incubated without pro-oxidants. However, exposure of sperm to more severe oxidative stress (500 mM H2O2 or a combination of 100 microM ascorbic acid, 20 microM FeSO4 and 500 microM H2O2) led to plasma membrane oxidation, reduced percentages of cleaved embryos and a reduction in the percentages of cleaved embryos that developed to the blastocyst stage. From these results, we conclude that the impact of oxidative stress to sperm becomes primarily manifest after the first cleavage of the formed zygote. Importantly, the level of lipid peroxidation in the sperm plasma membrane significantly correlates with blastocyst formation when the corresponding sperm is used for in vitro fertilization of oocytes.     

Restoration of Dry Afromontane Forest Using Pioneer Shrubs as Nurse-Plants for Olea europaea ssp. cuspidata

Shrubs are often considered competitive barriers for seedlings planted in reforestation programs, although they can facilitate tree recruitment, especially in ecosystems under high abiotic stress. An alternative reforestation technique using pioneer shrubs as nurse‐plants for Olea europaea ssp. cuspidata was tested in exclosures in northern Ethiopia. Seedlings were planted in three different microhabitats, and their survival was monitored. The microhabitats were bare soil patches between shrubs, patches under the dominant shrub Acacia etbaica, and patches under Euclea racemosa, an evergreen shrub, which supports the majority of naturally established Olea recruits. The ability of shrubs to offer protection against browsing goats was tested experimentally. Controlled shading was used to determine whether solar irradiation causes seedling mortality in environments without water stress. Data were analyzed using Kaplan–Meier survival analysis, Kruskal–Wallis analysis of variance (ANOVA), and one‐way ANOVA. Olea survival was significantly higher and shoot damage by goats was lower when planted under shrub cover compared to bare soil patches, particularly under Euclea canopies, although high shade levels reduced seedling performance. Reduction of solar radiation by shrub canopies and thus control of soil–water evaporation and seedling transpiration most likely controlled the observed facilitation. Planting under shrubs may increase seedling survival and assist regeneration of dry Afromontane vegetation. Preserving pioneers also reduces soil erosion and conserves biodiversity. Excluding livestock is essential for Olea woodland restoration and allows persistent but morphologically modified Olea shrubs to develop vigorous regrowth. Facilitative processes are guiding principles for assisted forest restoration, but above‐average rains may be critical to restore higher biomass levels in semiarid areas.     

A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin

Hemocyanins of mollusks are high molecular mass glycoproteins with a complex quaternary structure which still remains to be defined in detail for most of its species as far as number, spatial distribution and interactions of their structural units is concerned. In the present study, we isolated the functional units of the structural subunit RvH1 of Rapana venosa hemocyanin, combining enzymatic and non-enzymatic methods. Our results suggest that Hc's carbohydrate moieties play a basic role in the organization of the structural units, resulting from post-translational polymerization of the 50 kDa functional units and involving sugar moieties that link between them.