Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Structural perturbations induced by Asn131 and Asn171 glycosylation converge within the EFSAM core and enhance stromal interaction molecule-1 mediated store operated calcium entry.

A major intracellular calcium (Ca2+) uptake pathway in both excitable and non-excitable eukaryotic cells is store-operated Ca2+ entry (SOCE). SOCE is the process by which endoplasmic reticulum (ER)-stored Ca2+ depletion leads to activation of plasma membrane Ca2+ channels to provide a sustained increase in cytosolic Ca2+ levels that mediate a plethora of physiological processes ranging from the immune response to platelet aggregation. Stromal interaction molecule-1 (STIM1) is the principal regulator of SOCE and responds to changes in ER stored Ca2+ through luminal sensing machinery composed of EF-hand and SAM domains (EFSAM). The EFSAM domain can undergo N-glycosylation at Asn131 and Asn171 sites; however, the precise role of EFSAM N-glycosylation in the Ca2+ sensing mechanism of STIM1 is unclear. By establishing a site-specific chemical approach to covalently linking glucose to EFSAM and examining α-helicity, thermal stability, three dimensional atomic-resolution structure, Ca2+ binding affinity and oligomerization, we show that N-glycosylation of the EFSAM domain enhances the properties that promote STIM1 activation. This augmentation occurs through changes in structure localized near the Asn131 and Asn171 sites that together permeate through the protein core and lead to decreased Ca2+ binding affinity, reduced stability and enhanced oligomerization. Congruently, Ca2+ influx via SOCE in HEK293 cells co-expressing Orai1 and STIM1 was diminished when N-glycosylation was blocked by introducing Asn131Gln and Asn171Gln mutations. Collectively, our data suggests that N-glycosylation enhances the EFSAM destabilization-coupled oligomerization in response to ER Ca2+ depletion thereby augmenting the role of STIM1 as a robust ON/OFF regulator of SOCE. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Analysis of risk factors for lipid intolerance of intravenous fat emulsion in very low birth weight infants.

In order to prevent fatty acid deficiency and to supply enough energy, intravenous fat emulsion is necessary for parenteral nutrition in preterm neonates. However, parenteral administration of intravenous fat emulsion can induce lipid intolerance. The purpose of this study was to analyze risk factors for lipid intolerance in very low birth weight infants. This retrospective study included 80 preterm neonates whose birth weight was less than 1,500 g. Subjects were divided into 2 categories: those with a serum triglyceride level of ≥ 200 mg/dl (n = 33, 41%) and those with a serum triglyceride level of < 200 mg/dl (n = 47, 59%). We conducted logistic regression analysis using variables which were significant in univariate analysis. All statistical analyses were processed in SPSS version 19.0. Four risk factors for lipid intolerance were obtained through analysis of the electronic medical record. Lipid intolerance occurred more frequently in neonates with sepsis; those with a birth weight less than 1,000 g; those who was administered intravenous fat emulsion more than 2.6 g/kg/day; and those whose gestational age was less than 28 weeks. It is suggested that serum triglyceride levels should be closely monitored to prevent lipid intolerance in preterm neonates with the aforementioned characteristics.

Effect of lumbar stabilization and dynamic lumbar strengthening exercises in patients with chronic low back pain.

OBJECTIVE: To compare the effects of lumbar stabilization exercises and lumbar dynamic strengthening exercises on the maximal isometric strength of the lumbar extensors, pain severity and functional disability in patients with chronic low back pain (LBP). METHODS: Patients suffering nonspecific LBP for more than 3 months were included prospectively and randomized into lumbar stabilization exercise group (n=11) or lumbar dynamic strengthening exercise group (n=10). Exercises were performed for 1 hour, twice weekly, for 8 weeks. The strength of the lumbar extensors was measured at various angles ranging from 0° to 72° at intervals of 12°, using a MedX. The visual analog scale (VAS) and the Oswestry Low Back Pain Disability Questionnaire (ODQ) were used to measure the severity of LBP and functional disability before and after the exercise. RESULTS: Compared with the baseline, lumbar extension strength at all angles improved significantly in both groups after 8 weeks. The improvements were significantly greater in the lumbar stabilization exercise group at 0° and 12° of lumbar flexion. VAS decreased significantly after treatment; however, the changes were not significantly different between the groups. ODQ scores improved significantly in the stabilization exercise group only. CONCLUSION: Both lumbar stabilization and dynamic strengthening exercise strengthened the lumbar extensors and reduced LBP. However, the lumbar stabilization exercise was more effective in lumbar extensor strengthening and functional improvement in patients with nonspecific chronic LBP.

The assessment of efficacy of porcine reproductive respiratory syndrome virus inactivated vaccine based on the viral quantity and inactivation methods.

BACKGROUND: There have been many efforts to develop efficient vaccines for the control of porcine reproductive and respiratory syndrome virus (PRRSV). Although inactivated PRRSV vaccines are preferred for their safety, they are weak at inducing humoral immune responses and controlling field PRRSV infection, especially when heterologous viruses are involved. RESULTS: In all groups, the sample to positive (S/P) ratio of IDEXX ELISA and the virus neutralization (VN) titer remained negative until challenge. While viremia did not reduce in the vaccinated groups, the IDEXX-ELISA-specific immunoglobulin G increased more rapidly and to significantly greater levels 7 days after the challenge in all the vaccinated groups compared to the non-vaccinated groups (p < 0.05). VN titer was significantly different in the 106 PFU/mL PRRSV vaccine-inoculated and binary ethylenimine (BEI)-inactivated groups 22 days after challenge (p < 0.05). Consequently, the inactivated vaccines tested in this study provided weak memory responses with sequential challenge without any obvious active immune responses in the vaccinated pigs. CONCLUSIONS: The inactivated vaccine failed to show the humoral immunity, but it showed different immune response after the challenge compared to mock group. Although the 106 PFU/mL-vaccinated and BEI-inactivated groups showed significantly greater VN titers 22 days after challenge, all the groups were already negative for viremia.