One-step isolation and biochemical characterization of a highlyactive plant PSII monomeric core

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009)

Small molecule neuropilin-1 antagonists combine anti-angiogenic and anti-tumour activity with immune modulation through reduction of transforming growth factor beta (TGFβ) production in regulatory T-cells

We report the design, synthesis and comprehensive studybiological evaluation of a range ofsome potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumours, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229, EG00229 which was used a starting point for optimisation. Through targeting of specific amino-acid residues additional H-bonding interactions were introduced, which led to increases in binding affinity and potency. The design of these molecules was informed and supported by X-ray crystal structures. Pharmacokinetic data was obtained for some of the most potent compounds, and cCompound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays, and was shown to have anti-angiogenic, anti-migratory and anti-tumour effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, CD25+ populations of Tregs from mice 1 was able to block a glioma conditioned medium induced increase in TGFβ production. This study therefore represents a comprehensive characterisation of a small-molecule NRP1 antagonist, and provides the basis for future in vivo studies

The ATPase activities of sulfonylurea receptor 2A and sulfonylurea receptor 2B are influenced by the C-terminal 42 amino acids.

Unusually among ATP-binding cassette proteins, the sulfonylurea receptor (SUR) acts as a channel regulator. ATP-sensitive potassium channels are octameric complexes composed of four pore-forming Kir6.2 subunits and four regulatory SUR subunits. Two different genes encode SUR1 (ABCC8) and SUR2 (ABCC9), with the latter being differentially spliced to give SUR2A and SUR2B, which differ only in their C-terminal 42 amino acids. ATP-sensitive potassium channels containing these different SUR2 isoforms are differentially modulated by MgATP, with Kir6.2/SUR2B being activated more than Kir6.2/SUR2A. We show here that purified SUR2B has a lower ATPase activity and a 10-fold lower K(m) for MgATP than SUR2A. Similarly, the isolated nucleotide-binding domain (NBD) 2 of SUR2B was less active than that of SUR2A. We further found that the NBDs of SUR2B interact, and that the activity of full-length SUR cannot be predicted from that of either the isolated NBDs or NBD mixtures. Notably, deletion of the last 42 amino acids from NBD2 of SUR2 resulted in ATPase activity resembling that of NBD2 of SUR2A rather than that of NBD2 of SUR2B: this might indicate that these amino acids are responsible for the lower ATPase activity of SUR2B and the isolated NBD2 of SUR2B. We suggest that the lower ATPase activity of SUR2B may result in enhanced duration of the MgADP-bound state, leading to channel activation

Factors for thyroid autoimmunity in children and adolescents with type 1 diabetes mellitus

Introduction. Type 1 diabetes mellitus (T1DM) is associated with an autoimmune reaction to thyroid antigens including thyroid peroxidase (anti-TPO) and thyroglobulin (anti-Tg). Aims. We determined in children with T1DM the relationship of positive anti-thyroid antibodies to potential risk factors, including, age, gender, duration of diabetes, and glutamic acid decarboxylase antibodies (anti-GAD). Materials and methods. We studied 144 children and adolescents with T1DM. Their age was 12.3 ± 4.6 (mean ± SD) years, and duration of diabetes was 4.6 ± 3.8 years. Anti-thyroid antibodies were determined using a luminescence method and anti-GAD using an enzyme-linked immunosorbent assay. Results. The prevalence rates of anti-thyroid antibodies among the children with T1DM in our study were: anti-TPO (17.4%), anti-Tg (11.1%), and of both anti-thyroid antibodies (10.4%). The presence of serum anti-thyroid antibodies was positively associated with age (16.6 years in those with positive tests versus 12.0 years in those with negative tests, P 0.027), duration of diabetes (7.4 versus 4.3 years, P 0.031), and serum TSH (Thyroid-stimulating hormone) levels (4.8 versus 2.3 μIU/mL, P 0.002). The presence of both anti-thyroid antibodies was associated with female sex (boys: 4/75 (5.3%), girls: 11/69 (15.9%), chi-square 6.44, P 0.04). Subclinical autoimmune thyroiditis (SAIT) was present in 55.5% of the patients with thyroid antibody-positivity and was positively associated with age (16.6 versus 12.0 years, P 0.001) and diabetes duration (7.6 versus 4.2 years, P 0.001). Multiple logistic regression analysis revealed that the development of anti-thyroid antibodies was predicted by: 1) the presence of anti-GAD (odds ratio (OR) 1.45, 95% confidence interval (CI) 1.091.92), 2) the presence of a second anti-thyroid antibody (OR 134.4, 95% CI 7.72350.3), and 3) older age (OR 22.9, 95% CI 1.13463.2). Conclusions. Thyroid autoimmunity was associated with female gender, increasing age, long diabetes duration, the persistence of anti-GAD, and with TSH elevation, indicating subclinical hypothyroidism

Histological expression of angiogenic factors: VEGF, PDGFR alpha, and HIF-1 alpha in Hodgkin lymphoma

Angiogenesis is a prerequisite for solid tumor growth, but there is relatively limited data regarding Hodgkin lymphoma. The purpose of this study was to examine the immunohistochemical expression of angiogenic and proliferation markers in Hodgkin biopsies in relation to clinical parameters. Immunostaining was performed on 65 Hodgkin biopsies with vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1 alpha), platelet-derived growth factor receptor alpha (PDGFR alpha), Ki-67, and p53. Microvessel density (MVD) was determined by CD31 staining. In all cases, neoplastic cells and reactive background cells were evaluated. The neoplastic population expressed VEGF in 48% of the cases, HIF-1 alpha in 54% of the cases, and PDGFR alpha in 95% of the cases. Both Ki-67 and p53 were positive in neoplastic cells in over 60% of the cases. The MVD had a median of 2.6/0.0625 mm(2) which was not different from normal lymph nodes. VEGF in the non-neoplastic compartment showed increased staining in Ann Arbor stage I-II versus III-IV. In conclusion, VEGF, HIF-1 alpha, and predominantly PDGFRa are expressed in neoplastic cells in the majority of Hodgkin lymphomas. As microvessel formation is not increased in Hodgkin, additional functions of these angiogenic molecules should be investigated. (C) 2008 Elsevier GmbH. All rights reserved