Members of the Bunyaviridae family acquire their envelopes by budding into the Golgi complex (GC). The accumulation of the membrane glycoproteins G1 and G2 in the GC probably determines the site of maturation. Here we have studied the intracellular transport and targeting to the GC of G1 and G2 of Uukuniemi virus, a member of the Phlebovirus genus, and report on their expression from cloned cDNAs either together or separately by using a T7 RNA polymerase-driven vaccinia virus expression system. When G1 and G2 were expressed together from a full-length cDNA as the p110 precursor, both proteins were localized to the Golgi complex, as evidenced by colocalization with the Golgi marker enzyme mannosidase II. Immunofluorescent staining indicated that G1 expressed alone also localized to the GC. However, pulse-chase experiments showed that G1 remained endoglycosidase H sensitive. G2 expressed alone remained associated with the endoplasmic reticulum (ER). G2 could be rescued from the ER and transported to the GC by coexpression with G1 from separate mRNAs. Coexpression also increased the efficiency of G1 transport to the GC. With none of the constructs could the glycoproteins be observed on the cell surface. These results show that efficient export of G1 and G2 from the ER requires coexpression of both proteins, in conformity with our previous results showing that G1 and G2 form heterodimeric complexes in the ER. Since G1 expressed alone is retained in the GC, we conclude that G1 contains a retention signal for localization to the GC. G2 might thus become associated with the GC indirectly via its interaction with G1.

Background-Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, predicts mortality in cardiovascular disease and has been linked to cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). In this prospective study, we assessed whether circulating ADMA, arginine:ADMA ratio, and nitrite/nitrate levels were associated with survival and cerebral vasospasm in SAH patients. Methods-One hundred and eleven patients were observed day 1 to 15 after SAH, with serial measurements of transcranial Doppler flow velocities (V MCA ) and plasma biomarkers. Clinical status was assessed by the World Federation of Neurosurgical Societies grading scale. Results-Overall 30-day mortality was 18%, but differed between patients grouped by low, midrange, and high arginine:ADMA ratio in the first week after SAH. Mortality rates were 14/37, 1/37, and 5/37 in the 3 groups, respectively (P-logrank=0.0003). Cox regression showed that low versus midrange or high arginine:ADMA was associated with a hazard ratio of 4.1 independent of World Federation of Neurosurgical Societies grade (95% confidence interval, 1.5-10.9; P=0.006). ADMA or arginine:ADMA had no association to V MCA , but there was an inverse relationship between V MCA and nitrite/nitrate levels (P<0.0001). The NOS3 894G/G genotype was associated with 15% lower V MCA (P=0.01). ATbG-NOS3 haplotype homozygosity was associated with up to 64% higher nitrite/nitrate levels (P=0.003). Conclusions-This

Introduction: Hypoxia is associated with increased capillary permeability. This study tested whether acute hypobaric hypoxia involves degradation of the endothelial glycocalyx.Methods: We exposed 12 subjects to acute hypobaric hypoxia (equivalent to 4500 m for 2–4 h) and measured venous blood concentrations of biomarkers reflecting endothelial and glycocalyx degradation (catecholamines, syndecan-1, soluble CD40 ligand, protein C, soluble thrombomodulin, tissue-type plasminogen activators, histone-complexed DNA fragments, and nitrite/nitrate). Endothelial function was assessed by the hyperemic response to brachial artery occlusion by peripheral arterial tonometry.Results: Compared with normoxic baseline levels, hypoxia increased concentrations of syndecan-1 from 22 (95% confidence interval: 17–27) to 25 (19–30) ng/ml (p < 0.02) and protein C from 76 (70–83)% to 81 (74–88)% (p < 0.02). Nitrite/nitrate decreased from 23 (18–27) μM at baseline to 19 (14–24) μM and 18 (14–21) μM in hypoxia and recovery, respectively (p < 0.05). Other biomarkers remained unchanged. The post-occlusion/pre-occlusion ratio (reactive hyperemia index, RHI) decreased from 1.80 (1.52–2.07) in normoxia to 1.62 (1.28–1.96) after 2–4 h of hypobaric hypoxia and thereafter increased to 2.43 (1.99–2.86) during normoxic recovery (p < 0.01).Conclusions: The increase in syndecan-1 and protein C suggests that acute hypobaric hypoxia produces a minor degree of glycocalyx degradation and overall cellular damage. After hypoxia RHI rebounded to higher than baseline levels suggesting improved endothelial functionality.

Peripheral flow-mediated vasodilation is attenuated in the first days after SAH indicating acute systemic endothelial dysfunction. Impairment of endothelial function after SAH correlates with imbalance of the arginine/ADMA pathway.