Altered O-glycosylation of ß1-adrenergic receptor N-terminal single-nucleotide variants modulates receptor processing and functional activity.

N-terminal nonsynonymous single-nucleotide polymorphisms (SNPs) of G protein-coupled receptors (GPCRs) are common and often affect receptor post-translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human ß1-adrenergic receptor (ß1AR) is O-glycosylated in the N-terminal extracellular domain by polypeptide GalNAc transferase-2 that co-regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O-glycosylation assays, analysis of native receptor N-terminal O-glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O-glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O-glycosites that enhanced or inhibited cleavage at the adjacent sites (P52↓L53 and R31↓L32), respectively, or abolished the major site at R31↓L32 (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full-length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol-mediated signaling in an enhanced bystander bioluminescence energy transfer ß-arrestin2 recruitment assay in a coordinated manner with the common C-terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a ß1AR gain-of-function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.

GPR37 is processed in the N-terminal ectodomain by ADAM10 and furin.

GPR37 is an orphan G protein-coupled receptor (GPCR) implicated in several neurological diseases and important physiological pathways in the brain. We previously reported that its long N-terminal ectodomain undergoes constitutive metalloprotease-mediated cleavage and shedding, which have been rarely described for class A GPCRs. Here, we demonstrate that the protease that cleaves GPR37 at Glu167↓Gln168 is a disintegrin and metalloprotease 10 (ADAM10). This was achieved by employing selective inhibition, RNAi-mediated downregulation, and genetic depletion of ADAM10 in cultured cells as well as in vitro cleavage of the purified receptor with recombinant ADAM10. In addition, the cleavage was restored in ADAM10 knockout cells by overexpression of the wild type but not the inactive mutant ADAM10. Finally, postnatal conditional depletion of ADAM10 in mouse neuronal cells was found to reduce cleavage of the endogenous receptor in the brain cortex and hippocampus, confirming the physiological relevance of ADAM10 as a GPR37 sheddase. Additionally, we discovered that the receptor is subject to another cleavage step in cultured cells. Using site-directed mutagenesis, the site (Arg54↓Asp55) was localized to a highly conserved region at the distal end of the ectodomain that contains a recognition site for the proprotein convertase furin. The cleavage by furin was confirmed by using furin-deficient human colon carcinoma LoVo cells and proprotein convertase inhibitors. GPR37 is thus the first multispanning membrane protein that has been validated as an ADAM10 substrate and the first GPCR that is processed by both furin and ADAM10. The unconventional N-terminal processing may represent an important regulatory element for GPR37.

A dust-enshrouded tidal disruption event with a resolved radio jet in a galaxy merger.

Tidal disruption events (TDEs) are transient flares produced when a star is ripped apart by the gravitational field of a supermassive black hole (SMBH). We have observed a transient source in the western nucleus of the merging galaxy pair Arp 299 that radiated >1.5 × 1052 erg at infrared and radio wavelengths but was not luminous at optical or x-ray wavelengths. We interpret this as a TDE with much of its emission reradiated at infrared wavelengths by dust. Efficient reprocessing by dense gas and dust may explain the difference between theoretical predictions and observed luminosities of TDEs. The radio observations resolve an expanding and decelerating jet, probing the jet formation and evolution around a SMBH.

Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

The Parkinson's-disease-associated receptor GPR37 undergoes metalloproteinase-mediated N-terminal cleavage and ectodomain shedding.

The G-protein-coupled receptor 37 ( GPR37) has been implicated in the juvenile form of Parkinson's disease, in dopamine signalling and in the survival of dopaminergic cells in animal models. The structure and function of the receptor, however, have remained enigmatic. Here, we demonstrate that although GPR37 matures and is exported from the endoplasmic reticulum in a normal manner upon heterologous expression in HEK293 and SH-SY5Y cells, its long extracellular N-terminus is subject to metalloproteinase-mediated limited proteolysis between E167 and Q168. The proteolytic processing is a rapid and efficient process that occurs constitutively. Moreover, the GPR37 ectodomain is released from cells by shedding, a phenomenon rarely described for GPCRs. Immunofluorescence microscopy further established that although full-length receptors are present in the secretory pathway until the trans-Golgi network, GPR37 is expressed at the cell surface predominantly in the N-terminally truncated form. This notion was verified by flow cytometry and cell surface biotinylation assays. These new findings on the GPR37 N-terminal limited proteolysis may help us to understand the role of this GPCR in the pathophysiology of Parkinson's disease and in neuronal function in general.

Unfavourable short-term outcomes of a poly-L/D-lactide scaffold for thumb trapeziometacarpal arthroplasty.

The bioabsorbable poly-L-D-lactide joint scaffold arthroplasty is a recent attempt in the reconstruction of small joints in rheumatoid patients. In this study, we analysed the 1-year clinical, functional and radiologic results of partial trapeziectomy with the poly-L-D-lactide (96/4) joint scaffold in 23 patients with isolated trapeziometacarpal osteoarthritis. The results showed that the procedure provided pain relief and improvement in overall function according to the Quick Disabilities of the Arm, Shoulder and Hand score in most patients. However, radiographs demonstrated a high frequency of osteolysis around the implant. Seven patients developed clinically manifested foreign-body reactions 6 months to 1 year after surgery. The reason for the unexpected tissue reactions may relate to excessive mechanical cyclic loading of the implant. The outcomes of this implant in our patients have not been sufficiently beneficial and we have discontinued use of this implant in isolated trapeziometacarpal osteoarthritis.

Slowly fading super-luminous supernovae that are not pair-instability explosions.

Super-luminous supernovae that radiate more than 10(44) ergs per second at their peak luminosity have recently been discovered in faint galaxies at redshifts of 0.1-4. Some evolve slowly, resembling models of 'pair-instability' supernovae. Such models involve stars with original masses 140-260 times that of the Sun that now have carbon-oxygen cores of 65-130 solar masses. In these stars, the photons that prevent gravitational collapse are converted to electron-positron pairs, causing rapid contraction and thermonuclear explosions. Many solar masses of (56)Ni are synthesized; this isotope decays to (56)Fe via (56)Co, powering bright light curves. Such massive progenitors are expected to have formed from metal-poor gas in the early Universe. Recently, supernova 2007bi in a galaxy at redshift 0.127 (about 12 billion years after the Big Bang) with a metallicity one-third that of the Sun was observed to look like a fading pair-instability supernova. Here we report observations of two slow-to-fade super-luminous supernovae that show relatively fast rise times and blue colours, which are incompatible with pair-instability models. Their late-time light-curve and spectral similarities to supernova 2007bi call the nature of that event into question. Our early spectra closely resemble typical fast-declining super-luminous supernovae, which are not powered by radioactivity. Modelling our observations with 10-16 solar masses of magnetar-energized ejecta demonstrates the possibility of a common explosion mechanism. The lack of unambiguous nearby pair-instability events suggests that their local rate of occurrence is less than 6 × 10(-6) times that of the core-collapse rate.

Boar spermatozoa as a biosensor for detecting toxic substances in indoor dust and aerosols.

The presence, quantity and origins of potentially toxic airborne substances were searched in moisture damaged indoor environments, where building related ill health symptoms were suspected and reference sites with no health complaints. Boar spermatozoa were used as the toxicity sensor. Indoor aerosols and dusts were collected from kindergartens, schools, offices and residences (n=25) by electrostatic filtering, vacuuming, wiping from elevated surfaces and from the interior of personal computers. Toxicity was measured from the ethanol or methanol extracts of the dusts and aerosols. EC(50) was expressed as the lowest concentration of the airborne substance that inhibited motility of >50% of the exposed sperm cells compared to vehicle control, within 30 min, 1 day or 3-4 days of exposure. Remarkably toxic aerosols (EC(50)

A giant outburst two years before the core-collapse of a massive star.

The death of massive stars produces a variety of supernovae, which are linked to the structure of the exploding stars. The detection of several precursor stars of type II supernovae has been reported (see, for example, ref. 3), but we do not yet have direct information on the progenitors of the hydrogen-deficient type Ib and Ic supernovae. Here we report that the peculiar type Ib supernova SN 2006jc is spatially coincident with a bright optical transient that occurred in 2004. Spectroscopic and photometric monitoring of the supernova leads us to suggest that the progenitor was a carbon-oxygen Wolf-Rayet star embedded within a helium-rich circumstellar medium. There are different possible explanations for this pre-explosion transient. It appears similar to the giant outbursts of luminous blue variable stars (LBVs) of 60-100 solar masses, but the progenitor of SN 2006jc was helium- and hydrogen-deficient (unlike LBVs). An LBV-like outburst of a Wolf-Rayet star could be invoked, but this would be the first observational evidence of such a phenomenon. Alternatively, a massive binary system composed of an LBV that erupted in 2004, and a Wolf-Rayet star exploding as SN 2006jc, could explain the observations.

Malignancies after heart transplantation: presence of Epstein-Barr virus and cytomegalovirus.

The presence of Epstein-Barr virus (EBV), human papilloma virus (HPV), and cytomegalovirus (CMV) was studied in 20 patients who developed malignancies after heart transplantation in the Helsinki University Central Hospital. The tumors were analyzed for the presence of HPV by polymerase chain reaction and for EBV by in situ hybridization. Clinical CMV infection was verified by immunochemical quantitation of CMV antigen in peripheral blood cells. HPV was detected in one of the eight epithelial malignant tumors studied. Three of the six lymphomas were positive for EBV. Two (67%) of 3 patients with EBV-positive lymphomas and one (33%) of the other three lymphomas but only 2 (14%) of 14 patients who developed other malignancies had a history of a manifest post-transplantation CMV infection prior to the development of malignancy. These results confirm the presence of EBV in lymphomas of heart transplant recipients and suggest that CMV might have a contributory role in the development of EBV-associated lymphomas.

The effect of nitecapone on early graft function in experimental single lung transplantation.

Nitecapone is an antioxidant molecule which has been shown to protect the heart against ischemia-reperfusion injury. We investigated whether a similar effect could be detected on lung graft preservation in a porcine model of single lung transplantation. Donors received either nitecapone or placebo in a modified Euro-Collins pulmonary flush solution. After cold storage for 19 h the left lung was transplanted. Patients in the nitecapone group received a nitecapone infusion during the graft reperfusion. A right-side heart bypass was used to measure flow distribution and pulmonary vascular resistance (PVR) in the recipient's transplanted and native lungs, respectively. Pulmonary vein blood samples were analyzed for blood gases, free radical trapping capacity and diene conjugates. PVR was high in the transplanted lung, which received only 20% of the blood flow. Oxygen tension in the transplanted lung was low (2.3-26.7 kPa). Nitecapone treatment increased the plasma free radical trapping capacity threefold. In spite of this increase in antioxidative capacity nitecapone could not protect the lung against ischemia-reperfusion injury when pulmonary hemodynamics, gas exchange or plasma diene conjugates were used as measures of lung graft function.

Magnetic resonance imaging angiography in patency evaluation of bronchial artery revascularization grafts.

In this report we present our experience of non-invasive magnetic resonance imaging (MR) angiography and selective catheter angiography in assessing the patency of bronchial artery revascularization grafts after an en bloc double-lung and heart-lung transplantation. We studied 8 patients who had undergone pulmonary transplantation with direct bronchial artery revascularization. Catheter angiography was performed 10 days to 63 months postoperatively. MR angiography was performed within 24 h of the catheter procedure and the results were compared with the findings from catheter angiography. Catheter angiography showed the bronchial revascularization graft to be patent in 6 patients and occluded in 2. At MR angiography, the patency of bronchial artery revascularization grafts was reliably identified in 7 of the 8 patients. One patient had inadequate image quality because of void artefacts caused by haemostatic clips. It is concluded that MR angiography is a reliable method for assessing the patency of bronchial artery revascularization grafts.

Bronchial artery revascularization improves tracheal anastomotic healing after lung transplantation.

The study aimed to clarify the role of direct bronchial artery revascularization (BAR) after en bloc double-lung (DLT) and heart-lung transplantation (HLT). Group I comprised eight patients with en bloc DLT or HLT and successful BAR, while group II included 14 DLT or HLT cases without BAR or with failed BAR. From these groups, 2 subgroups were extracted: group III, including 6 cases of en bloc DLT with successful BAR and group IV 10 HLT cases without or with failed BAR. Airway healing was evaluated at bronchoscopy and patency of BAR with angiography. Pulmonary viral, bacterial and fungal infections, rejections and bronchiolitis obliterans syndrome (BOS) were registered. Tracheal healing at 2 weeks and 3 months was better in group I than in group 1 (p = 0.003 and p = 0.05, respectively). Compared with group IV, tracheal anastomotic healing at 2 weeks was better in group III (p = 0.007) and tended to be better also after 3 months (p = 0.07). The incidence of infections, rejection or BOS did not differ between groups I and II. BAR thus improved healing of tracheal anastomosis.

Major surgery increases serum levels of vascular endothelial growth factor only temporarily.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a hypoxia-induced endothelial cell-specific mitogen, which is angiogenic in vivo and up-regulated in several malignancies. VEGF can be used as a prognostic marker, but the effect of surgical trauma on serum VEGF (S-VEGF) concentrations is unknown and might reduce the value of VEGF as a serum marker. METHODS: We monitored S-VEGF levels by enzyme-linked immunosorbent assay in patients undergoing surgery. RESULTS: Eighteen patients with major surgery had slightly elevated S-VEGF compared with the preoperative level (median 9.5 pg/mL) on the first (median 35 pg/mL; P = 0.0002) and third (median 19 pg/mL; P = 0.004) postoperative day, but not in later samples. The levels measured in 8 patients after minor surgery did not differ from the preoperative levels (P = 0.14). CONCLUSIONS: Even major surgery is associated only with a slight and transient increase in S-VEGF levels, and, therefore, is unlikely to interfere markedly with the use of VEGF as a prognostic marker.

The role of pulmonary and systemic circulation in the tracheal blood supply in rats.

The different roles of bronchial and pulmonary circulation in the tracheal blood supply were investigated in 26 female rats: a control group (CG, n = 7), a group with pulmonary hilar ligation (PL, n = 5), another with tracheal transsection (TL, n = 9) and a group with both these procedures (TL&PL, n = 5). Technetium 99-m was injected into the left ventricle postoperatively, and the radioactivity of tracheal samples was calculated as a percentage of injected activity/g tissue (%ID/g). The tracheal uptake averaged 1.9 in group CG, and 1.7, 1.3 and 1.5% ID/g in groups PL, TL and TL&PL, respectively. Tracheal transsection (TL) thus reduced the tracheal blood supply by 29.7% compared with the control group (p < 0.05), whereas the reduction of tracheal blood supply following pulmonary hilar ligation (PL) was only 10.9% (n.s.). Tracheal transsection combined with hilar ligation (TL&PL) effected a reduction of 19.9% (n.s.). We conclude that only 10.9% of the tracheal blood supply comes from the pulmonary circulation.

Surgery for lung cancer in the elderly.

In order to assess the appropriateness of lung cancer surgery in the elderly and determine optimal subjects and resection procedure, 75 patients operated on in 1976-1996 at age > or =75 years (including 13 > or =80) were followed up. The operations included limited resection (8), lobectomy (47), bilobectomy (10) and pneumonectomy (10) and were judged to be radical in 59 cases (79%). Perioperative mortality was 9% and morbidity 29%, including 21% major complications. Cumulative 5-year survival was 32%, in stages IA-IIB 27-41%, and cancer-related survival 61-79%. Mortality did not differ significantly between resection types, but morbidity did. Nor did mortality, morbidity or survival differ between the age groups 75-79 and > or =80 years. In stage I cancer there was no significant difference in survival or cancer-related survival after lobectomy vs limited resection. We conclude that age, even >80 years, is not incompatible with curative resection. Lobectomy is the treatment of choice, but a less radical resection may be advisable if there is comorbidity. If more extensive resection is performed, the individual surgical risk must be weighed against the potential long-term benefit.

Donor lung pretreatment with prostaglandin E(1) does not improve lung graft preservation.

Prostaglandin E(1) (PGE(1)) is widely used to improve early graft function after lung transplantation, but some studies have questioned its benefits. Therefore we evaluated the effect of donor pretreatment with PGE(1) in our porcine model of single lung transplantation. Donors received PGE(1) or placebo intravenously before flushing the pulmonary artery with modified Euro-Collins solution. After cold storage, the excised left lung was transplanted. Ischemic time was 4 h. We used our right side heart bypass model to measure standardized pulmonary vascular resistance and to study blood flow distribution between recipient's native and transplanted lung. Systemic and pulmonary hemodynamics and gas exchange were also measured. After transplantation, pulmonary vascular resistance was significantly higher in the transplanted lung, which received only one fourth of the total pulmonary blood flow. PGE(1) pretreatment did not improve pulmonary hemodynamic parameters, or gas exchange.

Nitecapone inhibits myeloperoxidase in vitro and enhances functional performance after 8 h of ischemia in experimental heart transplantation.

Nitecapone (NC) has been shown to have beneficial effects on the functional recovery of rat hearts in Langendorff-preparation. The present study was executed to evaluate the effect of NC on preservation of grafts in heart transplantation and the role of NC in the inhibition of granulocyte infiltration. Donor hearts were perfused and stored at +4 degrees C for 8 h in either Ringer solution in the control-group (C-group, n = 26) or in NC (50 microM) added Ringer solution (NC-group, n = 18). The heterotopic heart transplantation was performed. The rats in both groups were killed at either 10 min or 60 min after release of the aortic clamp and tissue samples were obtained for antioxidative capacity, myeloperoxidase activity, and lipid peroxidation measurements. In vitro studies were performed using sodium azide or nitecapone to inhibit myeloperoxidase (MPO) activity of isolated human leukocytes. A total of 61% of the grafts began to beat in the NC-group, compared to 46% in the control group. Using an arbitrary scale of functional performance, only 33% (4/12) of the grafts were classified as well functioning in the control group, compared to 82% (9/11) in the NC-group (P<0.05). MPO activity was equal in both groups after 10 min but significantly lower after 60 min in the NC-group as compared to the control group (P<0.05). In vitro studies demonstrated that NC inhibits 50% of purified MPO activity at a concentration of 10 microM. NC did not significantly affect lipid peroxidation or the preservation of endogenous antioxidants. Since NC inhibited myeloperoxidase both in vitro and in vivo, it seems that the positive effects of NC on graft preservation may be mediated via the inhibition of granulocyte infiltration.