Protein kinase C epsilon is required for the induction of mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages

LPS induces in bone marrow macrophages the transient expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). Because MKP-1 plays a crucial role in the attenuation of different MAPK cascades, we were interested in the characterization of the signaling mechanisms involved in the control of MKP-1 expression in LPS-stimulated macrophages. The induction of MKP-1 was blocked by genistein, a tyrosine kinase inhibitor, and by two different protein kinase C (PKC) inhibitors (GF109203X and calphostin C). We had previously shown that bone marrow macrophages express the isoforms PKC beta I, epsilon, and zeta. Of all these, only PKC beta I and epsilon are inhibited by GF109203X. The following arguments suggest that PKC epsilon is required selectively for the induction of MKP-1 by LPS. First, in macrophages exposed to prolonged treatment with PMA, MKP-1 induction by LPS correlates with the levels of expression of PKC epsilon but not with that of PKC beta I. Second, G?6976, an inhibitor selective for conventional PKCs, including PKC beta I, does not alter MKP-1 induction by LPS. Last, antisense oligonucleotides that block the expression of PKC epsilon, but not those selective for PKC beta I or PKC zeta, inhibit MKP-1 induction and lead to an increase of extracellular-signal regulated kinase activity during the macrophage response to LPS. Finally, in macrophages stimulated with LPS we observed significant activation of PKC epsilon. In conclusion, our results demonstrate an important role for PKC epsilon in the induction of MKP-1 and the subsequent negative control of MAPK activity in macrophages.     

Colocalization of N-CAM and N-cadherin in avian skeletal myoblasts

The cell-cell adhesion molecules, N-CAM and N-cadherin, have been shown previously to mediate myoblast interaction during cell fusion accompanying skeletal myogenesis. To study the localization of both molecules in fusion-competent myoblasts, we used antigen-specific primary antibodies and a double-labeling preembedding immuno-electron microscopy technique. Ultrastructural observations and quantitative analysis of the results reveal that N-CAM and N-cadherin frequently colocalize in clusters on the myoblast plasma membrane. The data provide morphological evidence that the two adhesion glycoproteins cooperate in mediating myoblast interaction during myoblast fusion.     

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.     

Effect of HCO - 3 concentration in the absorption solution on the energetic coupling of H+-cotransports in roots of Zea mays L.

The effect of HCO ₃ ^- on ion absorption by young corn roots was studied in conditions allowing the independent control of both the pH of uptake solution and the CO₂ partial pressure in air bubbled through the solution. The surface pH shift in the vicinity of the outer surface of the plasmalemma induced by active H⁺ excretion was estimated using the initial uptake rate of acetic acid as a pH probe (Sentenac and Grignon (1987) Plant Physiol. 84, 1367). Acetic acid and orthophosphate uptake rates and NO ₃ ^- accumulation were slowed down, while ⁸⁶Rb⁺ uptake and K⁺ accumulation rates were increased by HCO ₃ ^- . These effects were similar to those induced by 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid/2-amino-2-(hydroxymethyl)-1,3-propanediol (Hepes-Tris). They were more pronounced when the H⁺ excretion was strong, were rapidly reversible and were not additive to those of Hepes-Tris. The hypothesis is advanced that the buffering system CO₂/H₂CO₃/HCO ₃ ^- accelerated the diffusion of equivalent H⁺ inside the cell wall towards the medium. This attenuated the surface pH shift in the vicinity the plasma membrane and affected the coupling between the proton pump and cotransport systems.Abbreviations FW fresh weight - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - J_aa acetic acid influx - J_K ⁺ K⁺ influx - J_Pi orthophosphate influx - Mes 2-(N-morpholino)ethanesulfonic acid - pCO₂ CO₂ partial pressure - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Long‐term coevolution between avian brood parasites and their hosts

Coevolutionary theory predicts that the most common long‐term outcome of the relationships between brood parasites and their hosts should be coevolutionary cycles based on a dynamic change selecting the currently least‐defended host species, given that when well‐defended hosts are abandoned, hosts will be selected to decrease their defences as these are usually assumed to be costly. This is assumed to be the case also in brood parasite‐host systems. Here I examine the frequency of the three potential long‐term outcomes of brood parasite–host coevolution (coevolutionary cycles, lack of rejection, and successful resistance) in 182 host species. The results of simple exploratory comparisons show that coevolutionary cycles are very scarce while the lack of rejection and successful resistance, which are considered evolutionary enigmas, are much more frequent. I discuss these results considering (i) the importance of different host defences at all stages of the breeding cycle, (ii) the role of phenotypic plasticity in long‐term coevolution, and (iii) the evolutionary history of host selection. I suggest that in purely antagonistic coevolutionary interactions, such as those involving brood parasites and their hosts, that although cycles will exist during an intermediate phase of the interactions, the arms race will end with the extinction of the host or with the host acquiring successful resistance. As evolutionary time passes, this resistance will force brood parasites to use previously less suitable host species. Furthermore, I present a model that represents the long‐term trajectories and outcomes of coevolutionary interactions between brood parasites and their hosts with respect to the evolution of egg‐rejection defence. This model suggests that as an increasing number of species acquire successful resistance, other unparasitized host species become more profitable and their parasitism rate and the costs imposed by brood parasitism at the population level will increase, selecting for the evolution of host defences. This means that although acceptance is adaptive when the parasitism rate and the costs of parasitism are very low, this cannot be considered to represent an evolutionary equilibrium, as conventional theory has done to date, because it is not stable.     

CCR7 expression and memory T cell diversity in humans

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.     

Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus

Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.     

Pathogenic bacteria and timing of laying

Pathogenic bacteria constitute a serious threat to viability of many organisms. Because growth of most bacteria is favored by humid and warm environmental conditions, earlier reproducers in seasonal environments should suffer less from the negative consequences of pathogenic bacteria. These relationships, and the effects on reproductive success, should be particularly prominent in predators because they are frequently exposed to pathogenic microorganisms from sick prey. Here, we presented and tested this hypothesis by sampling bacteria on adult and nestling goshawks Accipiter gentilis. We predicted that early breeders and their offspring should have fewer bacteria than those reproducing later during the breeding season. Adult goshawks with a high abundance of Staphylococcus on their beak and claws were easier to capture and their laying date was delayed. Moreover, goshawks that laid their eggs later had offspring with more Staphylococcus on their beaks and claws. The strength of the association between laying date and bacterial density of nestlings was stronger during the warm spring of 2013, when nestlings suffered from a higher abundance of pathogenic bacteria. Hatching failure and fledging failure were more common in nests with a higher abundance of Staphylococcus independently of the number of years occupied, laying date, and age of the female nest owner. These findings imply that timing of reproduction may be under the influence of pathogenic bacteria. Because early breeding goshawks produce more recruits than later breeders, our results suggest a role for pathogenic bacteria in the optimal timing of reproduction.     

Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5 degrees C in the epididymis for several days

The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.