Purification of glycomacropeptide from non-dialyzable fraction of sweet whey by anion-exchange chromatography

Glycomacropeptide (GMP) was purified from non-dialyzable fraction of sweet whey by anion-exchange chromatography on DEAE-Sephacel at two pHs 6.4 and 3.0. Chromatography at pH 3.0 (but not pH 6.4) gave a GMP fraction of high purity with its yield (1 g from every litre of whey) being approximately 100 times higher than that shown in the previous report. It was concluded that DEAE-Sephacel chromatography at pH 3.0 is a simple useful method to separate GMP from most whey proteins. It may be applicable to a large scale production of GMP.     

The effect of pre-eclamptic umbilical cord serum on fibroblast division in culture

Edema, proteinuria, hypertension (EPH-gestosis), most commonly termed as pre-eclampsia, is the most common pregnancy-associated pathological syndrome. It is accompanied by a thorough remodelling of extracellular matrix in the umbilical cord tissues. It is commonly known that the presence of serum in culture medium strongly stimulates many functions of cells cultured in vitro. It was decided to check how the pre-eclamptic serum affects the fibroblast division in culture. Ki-67 is a protein present in proliferating cells and can be detected during all phases of the cell cycle (G1, S, G2/M) but not in resting (G0) cells. PCNA (proliferating cell nuclear antigen) is an intranuclear polypeptide whose synthesis rate is at its maximum during the S-phase of the cell cycle. The expression of Ki-67 and PCNA was measured by immunocytochemical methods and biosynthesis of DNA was evaluated by [14C]-thymidine incorporation. The activity of pre-eclamptic umbilical cord serum (UC-serum) was found to be distinctly lower in comparison to control one. The expression of Ki and PCNA in fibroblast cultures treated with pre-eclamptic serum was also distinctly lower. Also the incorporation of [14C]-thymidine to DNA was lower than in the cultures treated with control UC-serum. It may by concluded that pre-eclampsia reduces the mitogenic activity of the umbilical cord serum.     

High serum sCD163/sTWEAK ratio is associated with lower risk of digital ulcers but more severe skin disease in patients with systemic sclerosis

Introduction

Systemic sclerosis (SSc) is an autoimmune disease characterized by chronic inflammation, vascular injury and excessive fibrosis. CD163 is a scavenger receptor which affects inflammatory response and may contribute to connective tissue remodelling. It has recently been demonstrated that CD163 can bind and neutralize the TNF-like weak inducer of apoptosis (TWEAK), a multifunctional cytokine which regulates inflammation, angiogenesis and tissue remodelling. We aimed to investigate the relationships between serum levels of soluble CD163 (sCD163) and soluble TWEAK (sTWEAK) in relation to disease manifestations in SSc patients.

Methods

This study included 89 patients with SSc who had not received immunosuppressive drugs or steroids for at least 6 months and 48 age- and sex-matched healthy controls (HC) from four European centres. Serum concentrations of sTWEAK and sCD163 were measured using commercially available ELISA kits.

Results

The mean serum concentrations of sTWEAK were comparable between SSc patients (mean +/- SD: 270 +/- 171 pg/mL) and HC (294 +/- 147pg/mL, P >0.05). Concentration of sCD163 and sCD163/sTWEAK ratio were significantly greater in SSc patients (984 +/- 420 ng/mL and 4837 +/- 3103, respectively) as compared to HC (823 +/- 331 ng/mL and 3115 +/- 1346 respectively, P <0.05 for both). High sCD163 levels and a high sCD163/sTWEAK ratio (defined as > mean +2SD of HC) were both associated with a lower risk of digital ulcers in SSc patients (OR, 95%CI: 0.09; 0.01, 0.71, and 0.17; 0.06, 0.51, respectively). Accordingly, patients without digital ulcers had a significantly higher sCD163 concentration and sCD163/sTWEAK ratio as compared to SSc patients with digital ulcers (P <0.01 for both) and HC (P <0.05 for both). A high sCD163/sTWEAK ratio, but not high sCD163 levels, was associated with greater skin involvement.

Conclusions

The results of our study indicate that CD163-TWEAK interactions might play a role in the pathogenesis of SSc and that CD163 may protect against the development of digital ulcers in SSc. Further studies are required to reveal whether targeting of the CD163-TWEAK pathway might be a potential strategy for treating vascular disease and/or skin fibrosis in SSc.     

Indole-3-acetic acid level in wood,bark and cambial sap of apple rootstocks differing in growth vigour

Free and conjugated IAA levels were determined in wood, bark and cambial sap of M.9, M.26 and MM.106 apple rootstock genotypes differing in growth vigour. The measurements were done on May 15^th, June 15^th and July 15^th. The level of free IAA in bark and wood of the tested trees varied from 27.0 to 52.7 ng·g⁻¹ f.w. while the conjugated hormone content averaged 3–5 times higher. In the bark and wood samples, the differences in auxin content between rootstock genotypes and the time of stem harvesting were insignificant. The level of free IAA in cambial sap was on average 10 to 20 times higher than in both bark and wood tissues, while the conjugated hormone level varied from none (below detection limit) to 37 ng·g⁻¹ f.w. Content of free IAA level in cambial sap from dwarf M.9 rootstock was significantly lower than that in either of the more vigorous genotypes. In both vigorous rootstocks IAA level in cambial sap remained at a similar level at all sampling dates but M.9 cambial sap showed a trend towards decreasing auxin content later in the growing season.     

47 Incorporation of alternative nucleic acid chemistries into G-quadruplex structure

Nucleic acids that form G-quadruplex (G4) structure have found applications in a host of research and technology regimes. Numerous G4 based aptamer drugs have been identified with pharmacological activity against cancer, HIV, prions, and blood coagulation (1). In the field of nanotechnology, G4 based sensors and nano-machines have also received much attention. The ability to synthesize nucleic acid ex-vivo allows for the site-specific incorporation of non-natural chemistries into nucleic acids that can be used to tune their physical and pharmacological properties. We summarize the results of a series of studies investigating the effective incorporation of alternative nucleic acid chemistries into G4 DNA. These modified chemistries include C8-modified guanine bases, as well as 2′-F, 2′-F-ANA, and Locked nucleic acid (LNA) modifications to the ribose sugar. We report primarily on the effect of these modifications on G-quadruplex folding topology, thermal stability, and structure. The substitution of LNA-guanosine into the core guanine tetrads disrupts structure in specific structural environments. On the other hand, 2′-F- and 2′-F-ANA guanosine can generally be incorporated without disrupting the structure when substituted into guanine bases in certain structural conformations. We find that 2′-F-ANA-guanosine and 2′-F-guanosine are powerful tools for controling the conformation of G4 structures (2). Functionalization at the C8 of the guanine base stabilizes in a manner dependent on the glycosidic conformation of the base, with different modification chemistries stabilizing to varying extents (3). The results of these studies provide useful insight on how to effectively incorporate some useful chemical tools from the growing toolbox of modified nucleic acid chemistries into G-quadruplex nucleic acid.     

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5 ng/ml. The detection limit is 0.06 ng/ml for the biosensor with antibody and 0.08 ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20 ng/ml for plasma samples and 0.30 to 0.49 ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.     

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.     

Alpha beta TCRs differ in the degree of their specificity for the positively selecting MHC/peptide ligand

We have tested the peptide specificity of positive selection using three transgenic alphabetaTCRs, originally selected on class II MHC (A(b)) covalently bound with one peptide Ealpha (52-68) (Ep). The transgenic TCR specific for the cytochrome c-derived (43-58) peptide was selected on A(b) bound with different arrays of endogenous peptides or the analogue of Ep covalently bound to A(b), but not on the original A(b)Ep complex. In contrast, transgenic TCRs specific for two different analogues of the Ep peptide and A(b) did not mature as CD4(+) T cells in various thymic environments, including the A(b)EpIi(-) mice. These results show that TCRs can be promiscuous or specific for the selecting MHC/peptide complex, and suggest that in mice described in this study transgenic expression of the TCR changes the original requirements for the positively selecting MHC/peptide complex. Future studies will determine whether the latter phenomenon is general or specific for this system.     

IGFs and IGF-Binding Proteins in the Synovial Fluid of Patients with Rheumatoid Arthritis and Osteoarthritis

International Journal of Peptide Research and Therapeutics - The progressive damage of human articular cartilage is associated with loss of integrity of its extracellular matrix components. Their...