Effect of juvenile hormone on caste determination and colony processes in the bumblebee Bombus terrestris

To investigate the role of juvenile hormone (JH) in caste determination, first and second instar larvae of Bombus terrestris were topically treated with one of three doses (2, 10, or 20 g/larva) of JH dissolved in acetone. Treated larvae belonged to very young colonies that had just been started by their queen. Therefore, all larvae were supposedly destined to develop into workers. Our study demonstrates that a single application of JH in the first or second instar can lead to the development of queens and that this effect is dose- and instar-dependent. The brood (second, or third brood of the colony) to which the larvae belonged also had an effect. A clear size dimorphism of the female castes exists in B. terrestris. In this study, however, intermediates also developed from treated larvae. In fact, even untreated larvae in a treated colony developed into queens and intermediates, depending on the total dose of JH applied to the colony. There are no indications that a larva, once determined to become a queen, can be forced to become a worker by means of malnutrition. Treatment with JH also had an influence on colony processes. For instance, the appearance of queen larvae resulted in the advanced start of reproduction by workers and egg robbery, the so called competition point. This indicates again the close relationship between queen rearing and the queen-worker conflict. However, the ultimate function of this casual connection is still unclear. Further, the queen reacted to the treatment by switching to the laying of haploid eggs at an earlier date in the colony development compared to untreated colonies.     

Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification

Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape of possible target sequences. The previous characterization of protein-DNA interaction before the engineering of new homing endonucleases is essential for further enzyme modification. Here we report the crystal structure of I-CvuI in complex with its target DNA and with the target DNA of I-CreI, a homologue enzyme widely used in genome engineering. To characterize the enzyme cleavage mechanism, we have solved the I-CvuI DNA structures in the presence of non-catalytic (Ca²⁺) and catalytic ions (Mg²⁺). We have also analyzed the metal dependence of DNA cleavage using Mg²⁺ ions at different concentrations ranging from non-cleavable to cleavable concentrations obtained from in vitro cleavage experiments. The structure of I-CvuI homing endonuclease expands the current repertoire for engineering custom specificities, both by itself as a new scaffold alone and in hybrid constructs with other related homing endonucleases or other DNA-binding protein templates.     

Sex steroid hormones do not influence the oxidative burst activity of polymorphonuclear leukocytes from ovariectomized cows in vitro

During the periparturient period, dairy cows are subjected to physiological changes that may induce immunosuppression and an increased susceptibility of the animal to bacterial infections such as mastitis. The incidence of clinical environmental mastitis is high during the last period of gestation, at parturition and during the first month of lactation, suggesting a potential influence of sex steroid hormones. Efficient functioning of polymorphonuclear leukocytes (PMN) is necessary during the early phase of infection to clear the mammary gland from invading pathogens. The purpose of this study was to investigate the effect of sex steroid hormones on the oxidative burst activity of isolated PMN from ovariectomized cows. Ovariectomy was performed to minimize the interference of endogenous estrogen and progesterone levels, which are known to vary extensively during the estrus cycle. Isolated PMN were incubated with different concentrations of 17beta-estradiol, estrone or progesterone. A flow cytometric technique was used to quantify the oxidation of intracellular 2',7'-dichlorofluorescin by the oxidative burst system of PMN following stimulation with phorbol myristate acetate. Staurosporine was used as a positive control for our in vitro model. No statistically significant changes in PMN oxidative burst activity were observed at physiological or pharmacological levels of the three sex steroid hormones. A large variation existed in the oxidative burst activity among cows. In an additional experiment, the expression of estrogen receptor alpha and of progesterone receptor in PMN was evaluated immunohistochemically. No specific staining was detected for both receptors in isolated PMN following incubation with different concentrations of sex steroid hormones.     

A phylogeny of Porella (Porellaceae, Jungermanniopsida) based on nuclear and chloroplast DNA sequences

The cosmopolitan family Porellaceae includes about 60 species in two or three genera: the large genus Porella and the monospecific Ascidiota and Macvicaria (alternatively Porella subg. Macvicaria). Maximum parsimony, maximum likelihood and Bayesian inference of phylogeny of a dataset including three markers (rbcL, trnL-trnF region of cp DNA, nrITS region) of 96 accessions resulted in similar topologies supporting the generic status of Ascidiota. Macvicaria is nested in a subclade of Porella. Relationships among species of Porella are in general well resolved and many terminal nodes achieve good statistical support whereas basal relationships are at best moderately supported. Multiple accessions of single species are usually placed in monophyletic lineages. Accessions of P. platyphylla split into a European and a North American clade with one accession from North America embedded within the European samples. The Macaronesian endemic P. inaequalis is closely related to the Asian species P. grandiloba. Porella obtusata and P. canariensis cannot be separated on the basis of the sequence data presented in this study. The molecular topologies indicate a range extension of the Asian P. gracillima subsp. urogea to Eastern North America and of the Neotropical P. swartziana to South Africa. Current supraspecific classifications of Porella are not reflected in the molecular topologies with a correlation between genetic variation and the geographical distribution of the related accessions rather than a correlation between genetic variation and morphology.     

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis

CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.     

Admission Lipoprotein-Associated Phospholipase A2 Activity Is Not Associated with Long-Term Clinical Outcomes after ST-Segment Elevation Myocardial Infarction

Background

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) activity is a biomarker predicting cardiovascular diseases in a real-world. However, the prognostic value in patients undergoing primary percutaneous coronary intervention (pPCI) for ST-segment elevation myocardial infarction (STEMI) on long-term clinical outcomes is unknown.

Methods

Lp-PLA₂ activity was measured in samples obtained prior to pPCI from consecutive STEMI patients in a high-volume intervention center from 2005 until 2007. Five years all-cause mortality was estimated with the Kaplan-Meier method and compared among tertiles of Lp-PLA2 activity during complete follow-up and with a landmark at 30 days. In a subpopulation clinical endpoints were assessed at three years. The prognostic value of Lp-PLA₂, in addition to the Thrombolysis In Myocardial Infarction or multimarker risk score, was assessed in multivariable Cox regression.

Results

The cohort (n = 987) was divided into tertiles (low <144, intermediate 144–179, and high >179 nmol/min/mL). Among the tertiles differences in baseline characteristics associated with long-term mortality were observed. However, no significant differences in five years mortality in association with Lp-PLA₂ activity levels were found; intermediate versus low Lp-PLA₂ (HR 0.97; CI 95% 0.68–1.40; p = 0.88) or high versus low Lp-PLA₂ (HR 0.75; CI 95% 0.51–1.11; p = 0.15). Both in a landmark analysis and after adjustments for the established risk scores and selection of cases with biomarkers obtained, non-significant differences among the tertiles were observed. In the subpopulation no significant differences in clinical endpoints were observed among the tertiles.

Conclusion

Lp-PLA₂ activity levels at admission prior to pPCI in STEMI patients are not associated with the incidence of short and/or long-term clinical endpoints. Lp-PLA₂ as an independent and clinically useful biomarker in the risk stratification of STEMI patients still remains to be proven.     

Analysis of a substrate specificity switch residue of cephalosporin acylase

Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( [Formula: see text] (cat)/ [Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of [Formula: see text] (m). The [Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain.     

Tangier disease: isolation and characterization of LpA-I,LpA-II,LpA-I:A-II and LpA-IV particles from plasma

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 ± 0.14 and 0.14 ± 0.04 μmol of cholesterol esterified/h/μg of protein, and 7 ± 2.5 and 1.4 ± 0.3 μmol of cholesteryl ester transferred/h/μg of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2–1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 ± 0.16 and 0.01 ± 0.01 μmol of cholesterol esterified/h/μg of protein and, 41 ± 3.7 and 1 ± 0.4 μmol of cholesteryl ester transferred /h/μg of protein). The LpA-II particle in TD represented in fact in LpA-II: A-IV complex (75% mol apoA-II and 22% mol apoA-IV). This particle did not promote cholesterol efflux, but LCAT and CETP activity were present. LpA-IV particles had the capacity to promote cholesterol efflux and had both LCAT and CETP activity. LpA-IV may contribute to maintain the reverse cholesterol transport in TD. Our results indicate the potential importance of apoA-IV in maintaining reverse cholesterol transport in TD. In spite of the low steady state HDL-cholesterol levels in TD, LpA-I, LpA-I: A-II: A-IV complex and LpA-IV appear to be active in reverse cholesterol transport and may help to prevent premature CHD in TD.     

Decreased Levels of Circulating IL17-Producing CD161+CCR6+ T Cells Are Associated with Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

The C-type lectin-like receptor CD161 is a well-established marker for human IL17-producing T cells, which have been implicated to contribute to the development of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). In this study, we analyzed CD161⁺ T cell recovery, their functional properties and association with GVHD occurrence in allo-SCT recipients. While CD161⁺CD4⁺ T cells steadily recovered, CD161^hiCD8⁺ T cell numbers declined during tapering of Cyclosporine A (CsA), which can be explained by their initial growth advantage over CD161^neg/lowCD8⁺ T cells due to ABCB1-mediated CsA efflux. Interestingly, occurrence of acute and chronic GVHD was significantly correlated with decreased levels of circulating CD161⁺CD4⁺ as well as CD161^hiCD8⁺ T cells. In addition, these subsets from transplanted patients secreted high levels of IFNγ and IL17. Moreover, we found that CCR6 co-expression by CD161⁺ T cells mediated specific migration towards CCL20, which was expressed in GVHD biopsies. Finally, we demonstrated that CCR6⁺ T cells indeed were present in these CCL20⁺ GVHD-affected tissues. In conclusion, we showed that functional CD161⁺CCR6⁺ co-expressing T cells disappear from the circulation and home to GVHD-affected tissue sites. These findings support the hypothesis that CCR6⁺CD161-expressing T cells may be involved in the immune pathology of GVHD following their CCL20-dependent recruitment into affected tissues.     

Sox2 is important for two crucial processes in lung development: branching morphogenesis and epithelial cell differentiation

The primary lung bud originates from the foregut and develops into the bronchial tree by repetitive branching and outgrowing of the airway. The Sry related HMG box protein Sox2 is expressed in a cyclic manner during initiation and branching morphogenesis of the lung. It is highly expressed in non-branching regions and absent from branching regions, suggesting that downregulation of Sox2 is mandatory for airway epithelium to respond to branch inducing signals. Therefore, we developed transgenic mice that express a doxycycline inducible Sox2 in the airway epithelium. Continuous expression of Sox2 hampers the branching process resulting in a severe reduction of the number of airways. In addition, the bronchioli transiently go over into enlarged, alveolar-like airspaces, a pathology described as bronchiolization of alveoli. Furthermore, a substantial increase was observed of cGRP positive neuroendocrine cells and ΔNp63 isoform expressing (pre-) basal cells, which are both committed precursor-like cells. Thus, Sox2 prevents airways from branching and prematurely drives cells into committed progenitors, apparently rendering these committed progenitors unresponsive to branch inducing signals. However, Sox2 overexpression does not lead to a complete abrogation of the epithelial differentiation program.