Characterization of chicken cystatin binding to rat renal brush-border membranes

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K_d = 3.67–4.07 μM) and high capacity (B_max = 2.32–2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K_i = 0.7 μM) among other megalin/cubilin ligands tested. The presence of Ca^+ 2 ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.     

Molecular basis of human CD36 gene mutations

CD36 is a transmembrane glycoprotein of the class B scavenger receptor family. The CD36 gene is located on chromosome 7 q11.2 and is encoded by 15 exons. Defective CD36 is a likely candidate gene for impaired fatty acid metabolism, glucose intolerance, atherosclerosis, arterial hypertension, diabetes, cardiomyopathy, Alzheimer disease, and modification of the clinical course of malaria. Contradictory data concerning the effects of antiatherosclerotic drugs on CD36 expression indicate that further investigation of the role of CD36 in the development of atherosclerosis may be important for the prevention and treatment of this disease. This review summarizes current knowledge of CD36 gene structure, splicing, and mutations and the molecular, metabolic, and clinical consequences of these phenomena.     

Visfatin levels do not change after the oral glucose tolerance test and after a dexamethasone-induced increase in insulin resistance in humans

INTRODUCTION, MATERIAL AND METHODS: Visfatin is a cytokine, mainly expressed in visceral fat, that exerts insulin-mimicking effects in rodents through activation of an insulin receptor, although the binding-site is distinct from that of insulin. However, the mechanisms that regulate visfatin synthesis are still not fully understood. In particular, it is not clear whether short-term glucose-induced hyperglycaemia and hyperinsulinaemia as well as a glucocorticoid-induced increase in insulin resistance are reflected in appreciable alterations in serum visfatin levels in humans. In order to investigate this we measured serum visfatin, glucose and insulin concentrations during a 75.0 gram oral glucose tolerance test (OGTT) [Study 1], as well as before and after oral administration of dexamethasone [Study 2]. Study 1 included 17 subjects (2 males), aged 35.7 +/- 15.6 (mean +/- SD) years of BMI 35.2 +/- 9.3 kg/m(2). Blood samples were taken before (0 minutes) and at 60 and 120 minutes after glucose administration. Study 2 included 20 subjects (4 males, 5 subjects with type 2 diabetes), aged 42.1 +/- 17.2 years of BMI 36.7 +/- 8.38 kg/m(2) who underwent screening for Cushing's disease/syndrome. Dexamethasone was administered at a dose of 0.5 mg every 6 hours for 48 hours. Fasting serum concentrations of visfatin, glucose and insulin were assessed before (D0) and after 48 hours of dexamethasone administration (D2). Insulin resistance was assessed according to the HOMA method in non-diabetic individuals (n = 15). RESULTS: In Study 1 two subjects were found to have impaired glucose tolerance and one subject was found to have diabetes mellitus. Glucose administration resulted in a highly significant increase in insulin (from 11.4 +/- 7.2 microU/mL at 0 min to 98.9 +/- 68.6 microU/mL at 60 min and 72.6 +/- 45.1 microU/mL at 120 minute of OGTT, p < 0.001 for 60 and 120 minutes in comparison to baseline). However, there was no change in serum visfatin concentrations (84.6 +/- 11.6 ng/mL at 0 minutes, 82.6 +/- 12.7 ng/mL at 60 minutes and 81.1 +/- 14.5 ng/mL at 120 minutes of OGTT, p = ns). All subjects in Study 2 achieved suppression of cortisol concentrations below 50 nmo/l. Dexamethasone administration resulted in an increase in fasting insulin (from 11.5 +/- 6.9 to 16.9 +/- 7.6 microU/mL; p = 0.011) and an increase in HOMA (from 2.73 +/- 1.74 to 4.02 +/- 2.27; p = 0.015), albeit without a significant change in serum visfatin concentrations (61.1 +/- 19.8 vs. 68.3 +/- 19.4 ng/mL, p = ns). In neither Study 1 nor Study 2 was there any significant correlation between serum visfatin and age, BMI or HOMA. CONCLUSIONS: There is a striking difference between the marked rise in insulin concentrations and the lack of change in visfatin concentrations during the oral glucose tolerance test. This implies that it is highly unlikely that visfatin is involved in the short-term regulation of glucose homeostasis in human subjects. Dexamethasone administration (4 mg/48 hours) induces an increase in insulin resistance, although without significant change in serum visfatin concentrations. Therefore in contrast to the in vitro data, short term glucocorticoid administration does not result in appreciable changes in serum levels of this adipocytokine. Furthermore, the results of our study do not support the notion that glucocorticoid-induced insulin resistance is likely to be related to changes in serum concentrations of visfatin.     

Prediction of protein secondary structure content for the twilight zone sequences

Secondary protein structure carries information about local structural arrangements, which include three major conformations: alpha-helices, beta-strands, and coils. Significant majority of successful methods for prediction of the secondary structure is based on multiple sequence alignment. However, multiple alignment fails to provide accurate results when a sequence comes from the twilight zone, that is, it is characterized by low (<30%) homology. To this end, we propose a novel method for prediction of secondary structure content through comprehensive sequence representation, called PSSC-core. The method uses a multiple linear regression model and introduces a comprehensive feature-based sequence representation to predict amount of helices and strands for sequences from the twilight zone. The PSSC-core method was tested and compared with two other state-of-the-art prediction methods on a set of 2187 twilight zone sequences. The results indicate that our method provides better predictions for both helix and strand content. The PSSC-core is shown to provide statistically significantly better results when compared with the competing methods, reducing the prediction error by 5-7% for helix and 7-9% for strand content predictions. The proposed feature-based sequence representation uses a comprehensive set of physicochemical properties that are custom-designed for each of the helix and strand content predictions. It includes composition and composition moment vectors, frequency of tetra-peptides associated with helical and strand conformations, various property-based groups like exchange groups, chemical groups of the side chains and hydrophobic group, auto-correlations based on hydrophobicity, side-chain masses, hydropathy, and conformational patterns for beta-sheets. The PSSC-core method provides an alternative for predicting the secondary structure content that can be used to validate and constrain results of other structure prediction methods. At the same time, it also provides useful insight into design of successful protein sequence representations that can be used in developing new methods related to prediction of different aspects of the secondary protein structure.     

Superoxide radical anions protect enkephalin from oxidation if the amine group is blocked

The pentapeptide methionine-enkephalin (Met-enk) is a natural opiate that inhibits signals of pain. The N-terminal tyrosyl residue is important in the recognition of the peptide by its receptor. In oxidative stress, this residue can be oxidized by reactive oxygen species. The one-electron oxidation of Met-enk and of tert-butoxycarbonyl-methionine-enkephalin (Boc-Met-enk) was studied by gamma- and pulse radiolysis in the absence and in the presence of superoxide radical anions (O(2)(.-)) and oxygen, using azidyl radicals as oxidants. Without oxygen, both peptides behaved similarly. The tyrosyl radical resulting from the oxidation of tyrosyl residue produced the dimer linked by dityrosines. Methionine was also oxidized to its sulfoxide; however, this reaction is of minor importance. When O(2)(.-) was present, it added to tyrosyl radical giving a hydroperoxide. For Met-enk, this adduct cyclized via an intramolecular Michael addition of the amine on the aromatic ring. Conversely, for Boc-Met-enk, the adduct eliminated oxygen which led to 97% regeneration of the nonmodified peptide. Blocking the terminal amine group had thus a key role in protection of the tyrosyl residue. This finding might be exploited in the search for new pain inhibitors.     

Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches

Background  

PD-(D/E)XK nucleases constitute a large and highly diverse superfamily of enzymes that display little sequence similarity despite retaining a common core fold and a few critical active site residues. This makes identification of new PD-(D/E)XK nuclease families a challenging task as they usually escape detection with standard sequence-based methods. We developed a modified transitive meta profile search approach and to consider the structural diversity of PD-(D/E)XK nuclease fold more thoroughly we analyzed also lower than threshold Meta-BASIC hits to select potentially correct predictions placed among unreliable or incorrect ones.     

Synthesis and biodegradation of nanogels as delivery carriers for carbohydrate drugs

Biodegradable nanogels loaded with rhodamine B isothiocyanate-dextran (RITC-Dx) as a model for water-soluble biomacromolecular drugs were prepared using atom-transfer radical polymerization (ATRP) in a cyclohexane inverse miniemulsion in the presence of a disulfide-functionalized dimethacrylate cross linker. UV-vis spectroscopy was used to characterize the extent of incorporation of RITC-Dx into the nanogels. The loading efficiency of RITC-Dx into the nanogels exceeded 80%. These nanogels were degraded into polymeric sols in a reducing environment to release the encapsulated carbohydrate drugs. The released carbohydrate biomolecules specifically interacted with concanavalin A in water, suggesting that the biodegradable nanogels could be used as carriers to deliver carbohydrate drugs that can be released upon degradation to bind to pathogens based on lectins.     

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.     

Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.