Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion

Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1–10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1–10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1–10 in analyses. The pre-maturated GFP 1–10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1–10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1–10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.Published versio

Novel Biochemical Markers of Psychosocial Stress in Women

Background: Prolonged psychosocial stress is a condition assessed through self-reports. Here we aimed to identify biochemical markers for screening and early intervention in women

Minimum information about a protein affinity reagent (MIAPAR)

This is a proposal developed within the community as an important first step in formalizing standards in reporting the production and properties of protein binding reagents, such as antibodies, developed and sold for the identification and detection of specific proteins present in biological samples. It defines a checklist of required information, intended for use by producers of affinity reagents, qualitycontrol laboratories, users and databases. We envision that both commercial and freely available affinity reagents, as well as published studies using these reagents, could include a MIAPAR-compliant document describing the product’s properties with every available binding partner. This would enable the user or reader to make a fully informed evaluation of the validity of conclusions drawn using this reagent

Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection

The introduction of different methodologies for construction and screening ofcomplex protein libraries has provided powerful means in protein engineeringfor development of molecules with desired traits. A challenge faced in manysituations is to adapt a given methodology for efficient and rapid identification ofthe most interesting variants present in a library. In the present study, theconcept of Darwinian selection based on a growth advantage for clones havingthe desired trait has been investigated. Using a β-lactamase-based ProteinFragment Complementation Assay (PCA), an affinity maturation of a TNF-αbinding affibody molecule of an initial 2 nM affinity for the target has beenperformed. Initial characterization of the PCA system, based on the affinitydriven reconstitution of β-lactamase activity in the periplasm of cells harbouringa library member showing affinity for a co-expressed target protein, showed thatthe system was responsive to promoter induction level, interaction affinity andapplied selection pressure. Using combinatorial protein engineering principles, a107 library of second generation affibody molecules was constructed andsubjected to selection of improved variants by library growth in liquid culture.The results showed that after a pre-selection step on semi-solid media toeliminate non-binding variants, present in majority, two rounds of selection inliquid culture resulted in an enrichment for binders showing up ten-fold higheraffinity to the TNF-α target than the ancestral variant. Biosensor analysesshowed that the major factor for the improved affinity could be attributed toreduced off-rate constants.Uppdaterad från manuskript till artikel: 20100729QC 2010072

Sjoholm A. Persistent endothelial dysfunction is related to elevated C-reactive protein (CRP) levels in Type II diabetic patients after acute myocardial infarction. Clin Sci

A B S T R A C T The atherosclerotic process is an ongoing dynamic and progressive state arising from endothelial dysfunction and inflammation. Although suffering from an acute coronary artery disease, patients with Type II diabetes have a poor outcome compared with non-diabetic patients, which may only partly be explained by traditional risk factors. Our purpose was to compare non-traditional risk factors, such as endothelial function, C-reactive protein (CRP) and adiponectin, in Type II diabetic and non-diabetic patients following AMI (acute myocardial infarction). Twenty Type II diabetic patients were compared with 25 non-diabetic patients at baseline (1-3 days from the onset of chest pain) and at 60 days follow-up after an AMI. Using high-resolution ultrasound, brachial artery responses to FMD (flow-mediated vasodilatation; endothelium-dependent vasodilatation) and NTG (nitroglycerine-induced vasodilatation; endothelium-independent vasodilatation) were measured. Plasma levels of CRP and adiponectin were measured by ELISA. At baseline, FMD (1.9 compared with 3.2 %; P = 0.22) and CRP levels (6.95 compared with. 5.51 mg/l; P = 0.40) did not differ between Type II diabetic and non-diabetic patients, whereas adiponectin levels were lower in Type II diabetic patients (2.8 compared with 5.0 ng/ml; P < 0.05). At 60 days followup, there were significant differences in FMD (1.5 compared with 4.1 %; P < 0.02), CRP (4.23 compared with 1.46 mg/ml; P < 0.01) and adiponectin (3.3 compared with 5.3 ng/ml; P < 0.05) levels between Type II diabetic and non-diabetic patients. In contrast, NTG responses improved in both groups between baseline and follow-up (Type II diabetic patients, 9.7 compared with 13.2 % respectively, P < 0.05; non-diabetic patients, 7.9 compared with 12.4 % respectively, P < 0.01). These results show a persistent endothelium-dependent dysfunction and inflammatory activity in patients with Type II diabetes, but not in non-diabetic patients, after AMI. These findings may, in part explain, the poor outcome in coronary artery disease seen in Type II diabetes

Quality of care in geriatric rehabilitation: Clients’ perceptions, ADL dependence, and subjective well-being in a one-year perspective.

World-wide, the development of community-based geriatric rehabilitation has received increased attention. In Sweden, national reforms during the 1990s aimed at improved quality of geriatric rehabilitation. This paper focuses clients' perceptions of the rehabilitation process, dependence in activities of daily living (ADL), and subjective well-being in a one-year perspective. A study-specific questionnaire, a revised version of the ADL Staircase, and the Göteborg Quality of Life Instrument were administered, in 1996 (N = 278) and 1997 (N = 233). Even if 77% of the clients were content as regards rehabilitation quality, in 1997 contentment diminished among clients in sheltered housing facilities. Most clients also reported a diminished contentment with the training provided during the period investigated. Most clients were dependent in ADL, but in sub-groups independence in some activities diminished over the study period. In contrast, in some aspects sub-groups scored their subjective well-being lower on the second measurement than on the first. The investigation of clients' perceptions of quality of care is a multifaceted matter, and the results of this study were partly ambiguous. Still, since valid descriptions of variables at target for rehabilitation is one important key to the continuous process of quality development, this study produced information valuable for further studies following geriatric rehabilitation processes over time. The implementation of this study could be applicable in similar settings. Read More: http://informahealthcare.com/doi/abs/10.1080/11038120175046450