Does nitric oxide act in the ventromedial preoptic area to mediate oestrogen negative feedback in the seasonally anoestrous ewe?

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containing Nomega-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA. l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor), l-NAME, SMTC, or empty implants. 7NI, but not l-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections of l-NAME were compared in mid-anoestrus. Microinjections of l-NAME (300 nl at 10 microg/microl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1-3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection of l-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.     

Identification of Candida albicans wall mannoproteins covalently linked by disulphide and/or alkali‐sensitive bridges

This paper describes the results obtained by analysing the human pathogen Candida albicans cell wall subproteome by mass spectrometry, using extraction procedures aimed at releasing proteins bound by disulphide bridges (RAE‐CWP) or alkali‐labile ester linkages (ALS‐CWP). Ten of the total proteins released from the wall by β‐ME and/or NaOH contained a potential signal peptide, lacked a GPI cell wall hydrophobic C‐terminal domain and were identified as true wall proteins by in silico analysis, whereas four additional proteins were identified as bound to the plasma membrane. The results surprisingly demonstrated that, in addition to the expected RAE‐CWP and ALS‐CWP proteins, 16 GPI proteins were bound to the wall by disulphide or alkali‐sensitive bonds, since they were released by β‐ME and/or NaOH. The biological significance of these results is discussed in relation to the added complexity of the organization of the proteins in the C. albicans cell wall. Copyright © 2014 John Wiley & Sons, Ltd.     

Sustainable Production of Levulinic Acid from the Cellulosic Fraction of Pinus Pinaster Wood: Operation in Aqueous Media Under Microwave Irradiation

The fractionation of the structural components of lignocellulosic biomass (cellulose, hemicelluloses, and lignin) and the separate utilization of the resulting fractions for specific purposes, according to the philosophy of biorefineries, enables the development of sustainable processes for biomass utilization. In this work, Pinus pinaster wood was subjected to aqueous processing to remove water-soluble extractives and hemicelluloses, and the resulting solid was subjected to pulping with HCl-catalyzed acetic acid solutions (Acetosolv method). The pulp was employed as a substrate for levulinic acid manufacture by reaction in acidic media under microwave irradiation. The effects of the major operational variables (temperature, reaction time, and acid concentration) on the levulinic acid yield were established by statistical modeling of experimental data. Operating under the best reaction conditions (at 191.2°C for 18.9 min in aqueous media containing 1.10% HCl), the levulinic acid yield accounted for 56.4% of the stoichiometric value.     

Diagnosis,Classification and Management of Mast Cell Activation Syndromes (MCAS) in the Era of Personalized Medicine

Mast cell activation (MCA) is seen in a variety of clinical contexts and pathologies, including IgE-dependent allergic inflammation, other immunologic and inflammatory reactions, primary mast cell (MC) disorders, and hereditary alpha tryptasemia (HAT). MCA-related symptoms range from mild to severe to life-threatening. The severity of MCA-related symptoms depends on a number of factors, including genetic predisposition, the number and releasability of MCs, organs affected, and the type and consequences of comorbid conditions. In severe systemic reactions, MCA is demonstrable by a substantial increase of basal serum tryptase levels above the individual’s baseline. When, in addition, the symptoms are recurrent, involve more than one organ system, and are responsive to therapy with MC-stabilizing or mediator-targeting drugs, the consensus criteria for the diagnosis of MCA syndrome (MCAS) are met. Based on the etiology of MCA, patients can further be classified as having i) primary MCAS where KIT-mutated, clonal, MCs are detected; ii) secondary MCAS where an underlying IgE-dependent allergy or other reactive MCA-triggering pathology is found; or iii) idiopathic MCAS, where neither a triggering reactive state nor KIT-mutated MCs are identified. Most severe MCA events occur in combined forms of MCAS, where KIT-mutated MCs, IgE-dependent allergies and sometimes HAT are detected. These patients may suffer from life-threatening anaphylaxis and are candidates for combined treatment with various types of drugs, including IgE-blocking antibodies, anti-mediator-type drugs and MC-targeting therapy. In conclusion, detailed knowledge about the etiology, underlying pathologies and co-morbidities is important to establish the diagnosis and develop an optimal management plan for MCAS, following the principles of personalized medicine.     

Stem cell factor-induced downregulation of c-kit in human lung mast cells and HMC-1 mast cells

Recent data suggest that local overexpression of the tissue-hormone c-kit ligand (stem cell factor [SCF]) is associated with accumulation of mast cells (MCs) and a decrease in expression of c-kit in the accumulated MCs [28]. In the present study, the effects of recombinant human (rh) SCF on expression of c-kit mRNA and c-kit protein in isolated human MCs and a human mast cell line, HMC-1, were analyzed. Incubation of isolated lung MC with rhSCF (100 ng/mL) for 120 minutes resulted in decreased expression of c-kit mRNA (optical density [OD], control: 100% vs. rhSCF: 37%). Almost identical results were obtained with HMC-1 cells (OD, control: 100% vs. rhSCF: 40 to 45%). As assessed by flow cytometry and monoclonal antibodies (mAbs) to c-kit, the SCF-induced decrease of c-kit mRNA in HMC-1 was associated with a substantial decrease in surface expression of c-kit (MFI, control: 100 +/- 21%, vs. MFI in cells incubated with rhSCF [100 ng/mL at 37 degrees C for 12 hours]: 8 +/- 2%, vs. MFI in cells incubated with rhSCF, 100 ng/mL, at 4 degrees C: 34 +/- 3%). The effects of rhSCF on c-kit expression in HMC-1 cells were dose- and time-dependent with maximum effects observed with 10-100 ng/mL of rhSCF after 4 to 12 hours. The SCF-dependent loss of c-kit was also accompanied by a decreased chemotactic response to rhSCF (control: 100%; rhSCF: 71 +/- 2%). This study shows that exposure of human lung MC and HMC-1 cells to recombinant SCF results in downregulation of c-kit mRNA and surface c-kit expression. These data may explain the partial loss of c-kit on MCs in areas of SCF overexpression.     

Cyclical etidronate in the treatment of postmenopausal osteoporosis: efficacy and safety after seven years of treatment

PURPOSE: To determine the efficacy and safety of cyclical etidronate for up to 7 years in the treatment of postmenopausal osteoporosis and to examine the effects of discontinuing treatment after 2 or 5 years of therapy. PATIENTS AND METHODS: Patients were randomized at entry into the original study in 1986 to blinded treatment for 2 years with either a calcium (placebo) or an intermittent cyclical etidronate regimen, which most patients continued for a third year. Following this phase of the study, patients were enrolled into an open-label, follow-up study (years 4 and 5), during which all patients received cyclical etidronate treatment. In the present double-blind study (years 6 and 7), patients were rerandomized to receive intermittent cyclical therapy with either etidronate or placebo; all patients received calcium. The treatment regimen consisted of 400 mg/day etidronate or placebo for 14 days, followed by 76 days of elemental calcium (500 mg/day); this cycle was repeated approximately 4 times in each year. Of the 193 patients who continued in years 6 and 7 of the study, 93 were randomized to receive cyclical etidronate and 100 were randomized to receive calcium only. For purposes of efficacy analyses, patients were categorized by their total years of cumulative etidronate treatment (7, 5, 4, or 2 years). There were 51, 46, 42, and 54 patients in the 7-, 5-, 4-, and 2-year groups, respectively. Annual assessments included lumbar spine bone mineral density (BMD), as measured by densitometry, and vertebral radiographs. RESULTS: The groups receiving cyclical etidronate during this 2-year study period (7- and 4-year groups) had statistically significant mean percent increases in spinal BMD of 1.8% and 2.2%, respectively (P < 0.05) at the week 104 observation time. The 5- and 2-year groups, which did not receive etidronate during this period, had mean values of 1.4% and 0.2%, respectively (not significant) at week 104. In the 7-, 5-, 4-, and 2-year groups, the increases in spinal BMD at the end of 7 years were 7.6%, 8.6%, 8.1%, and 3.9%, respectively; these values were statistically significant for all groups compared with original baseline (year 0) (P < 0.05). BMD of the femur and wrist was maintained throughout the 7-year period. The incidence and rate of vertebral fractures were lowest in patients with the longest exposure to etidronate. Etidronate was well tolerated during the study, with low incidences of gastrointestinal side effects and nonvertebral fractures. CONCLUSIONS: Long-term cyclical etidronate is a safe, effective, and well-tolerated treatment for postmenopausal osteoporosis. Bone mass is maintained for at least 2 years after treatment with etidronate is stopped; however, further gains in spinal bone mass are seen in patients who continue therapy.     

Ibuprofen inhibits pyrogen-dependent expression of VCAM-1 and ICAM-1 on human endothelial cells

Leukocyte adhesion and transmigration through the endothelial cell (EC) layer plays a crucial role in inflammation. IL-1 alpha and TNF alpha increase EC-adhesiveness for leukocytes by stimulating surface expression of ICAM-1 (intercellular adhesion molecule 1, CD54), VCAM-1 (vascular cell adhesion molecule 1, CD106) and E-selectin (CD62E). In this study, the effects of ibuprofen on IL-1 alpha and TNF alpha-induced expression of ICAM-1, VCAM-1 and E-selectin on cultured human umbilical vein EC (HUVEC) were analyzed. Exposure to IL-1 alpha or TNF alpha resulted in an increased expression of VCAM-1, ICAM-1, and E-selectin. Ibuprofen was identified as a potent inhibitor of IL-1 alpha and TNF alpha-induced surface expression of VCAM-1 and a less potent inhibitor of pyrogen-induced expression of ICAM-1, whereas no effect on E-selectin was found. The effects of ibuprofen on VCAM-1 expression were dose-dependent (IC50 [IL-1 alpha]: 0.5 mM; IC50 [TNF alpha]: 0.5 mM) and time-dependent with maximum responses observed after 18 h. Moreover, ibuprofen abrogated pyrogen-dependent adhesion of leukocytes to HUVEC. Ibuprofen also inhibited VCAM-1 mRNA expression in pyrogen activated EC. VCAM-1-downregulation on EC by ibuprofen may contribute to the anti-inflammatory actions of the drug.     

Chloroplast SRP54 interacts with a specific subset of thylakoid precursor proteins

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.     

The Displacement Calculus

If all dependent expressions were adjacent some variety of immediate constituent analysis would suffice for grammar, but syntactic and semantic mismatches are characteristic of natural language; indeed this is a, or the, central problem in grammar. Logical categorial grammar reduces grammar to logic: an expression is well-formed if and only if an associated sequent is a theorem of a categorial logic. The paradigmatic categorial logic is the Lambek calculus, but being a logic of concatenation the Lambek calculus can only capture discontinuous dependencies when they are peripheral. In this paper we present the displacement calculus, which is a logic of intercalation as well as concatenation and which subsumes the Lambek calculus. On the empirical side, we apply the new calculus to discontinuous idioms, quantification, VP ellipsis, medial extraction, pied-piping, appositive relativisation, parentheticals, gapping, comparative subdeletion, cross-serial dependencies, reflexivization, anaphora, dative alternation, and particle shift. On the technical side, we prove that the calculus enjoys Cut-elimination.