Selective neoglycosylation increases the structural stability of vicilin, the 7S storage globulin from pea seeds

The effects of glycosylation on the stability and subunit interactions of vicilin, the major storage protein in pea seeds, were investigated. Glycosylated vicilin derivatives were prepared by alkylation of lysine epsilon-amino groups with various carbohydrates. Average modification levels of 13.4 +/- 3.0, 11.1 +/- 3.6, 7.5 +/- 4.2, and 4.7 +/- 0.3 moles of carbohydrate/mol of vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046-4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (deltaG(diss)) and molar volume change (deltaV(diss)) of dissociation of vicilin subunits. For unmodified vicilin, deltaG(diss) = 18.2 kcal/mol and deltaV(diss) = -102 ml/mol. Glycosylated vicilin derivatives were significantly stabilized against subunit dissociation, with deltaG(diss) of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in deltaV(diss) were found for the glucose and galactose derivatives, whereas deltaV(diss) of -128 and -135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified vicilin. These results indicate that the stability and subunit interactions of vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.     

Tip off the HAT– Epigenetic control of learning and memory by Drosophila Tip60

Disruption of epigenetic gene control mechanisms involving histone acetylation in the brain causes cognitive impairment, a debilitating hallmark of most neurodegenerative disorders. Histone acetylation regulates cognitive gene expression via chromatin packaging control in neurons. Unfortunately, the histone acetyltransferases (HATs) that generate such neural epigenetic signatures and their mechanisms of action remain unclear. Our recent findings provide insight into this question by demonstrating that Tip60 HAT action is critical for morphology and function of the mushroom body (MB), the learning and memory center in the Drosophila brain. We show that Tip60 is robustly produced in MB Kenyon cells and extending axonal lobes and that targeted MB Tip60 HAT loss results in axonal outgrowth disruption. Functional consequences of loss and gain of Tip60 HAT levels in the MB are evidenced by defects in memory. Tip60 ChIP-Seq analysis reveals enrichment for genes that function in cognitive processes and accordingly, key genes representing these pathways are misregulated in the Tip60 HAT mutant fly brain. Remarkably, increasing levels of Tip60 in the MB rescues learning and memory deficits resulting from Alzheimer''s disease associated amyloid precursor protein (APP) induced neurodegeneration. Our studies highlight the potential of HAT activators as a therapeutic option for cognitive disorders.     

Modification of the detrimental effect of TNF‐α on human endothelial progenitor cells by fasudil and Y27632

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor‐α (TNF‐α) reduced their number and biological activity. Yet, signal transduction events linked to TNF‐α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in‐ flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose‐dependent improvement of EPC number during exposure to TNF‐α (P < 0.05 vs. TNF‐α alone). Analysis of the signal transduction pathway activated by TNF‐α revealed that the increased expression of p‐p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF‐α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF‐α) as well as the inhibitor of Erk1/2‐specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF‐α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D‐DIGE and MALDI‐TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin‐related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:351–360, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20345     

Isolation of an <Emphasis Type="Italic">Escherichia coli</Emphasis> K4 <Emphasis Type="Italic">kfoC</Emphasis> mutant over-producing capsular chondroitin

Background  

Chondroitin sulphate is a complex polysaccharide having important structural and protective functions in animal tissues. Extracted from animals, this compound is used as a human anti-inflammatory drug. Among bacteria, Escherichia coli K4 produces a capsule containing a non-sulphate chondroitin and its development may provide an efficient and cheap fermentative production of the polysaccharide.     

Modular organization of the Sulfolobus solfataricus mini-chromosome maintenance protein

Mini-chromosome maintenance (MCM) proteins form ring-like hexameric complexes that are commonly believed to act as the replicative DNA helicase at the eukaryotic/archaeal DNA replication fork. Because of their simplified composition with respect to the eukaryotic counterparts, the archaeal MCM complexes represent a good model system to use in analyzing the structural/functional relationships of these important replication factors. In this study the domain organization of the MCM-like protein from Sulfolobus solfataricus (Sso MCM) has been dissected by trypsin partial proteolysis. Three truncated derivatives of Sso MCM corresponding to protease-resistant domains were produced as soluble recombinant proteins and purified: the N-terminal domain (N-ter, residues 1-268); a fragment comprising the AAA+ and C-terminal domains (AAA+-C-ter, residues 269-686); and the C-terminal domain (C-ter, residues 504-686). All of the purified recombinant proteins behaved as monomers in solution as determined by analytical gel filtration chromatography, suggesting that the polypeptide chain integrity is required for stable oligomerization of Sso MCM. However, the AAA+-C-ter derivative, which includes the AAA+ motor domain and retains ATPase activity, was able to form dimers in solution when ATP was present, as analyzed by size exclusion chromatography and glycerol gradient sedimentation analyses. Interestingly, the AAA+-C-ter protein could displace oligonucleotides annealed to M13 single-stranded DNA although with a reduced efficiency in comparison with the full-sized Sso MCM. The implications of these findings for understanding the DNA helicase mechanism of the MCM complex are discussed.     

Molecular analysis of a novel tandemly organized repetitive DNA sequence in<Emphasis Type="Italic">Citrus limon</Emphasis> (L.) Burm

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and molecular analysis of a novel tandemly organized repetitive DNA sequence from the genome of Citrus limon. Digestion of C. limon DNA with Hinf I produced a prominent fragment of approximately 300 bp. Southern blotting revealed a ladder composed of DNA fragments that were multimers of the 300-bp Hinf I band. Thus, Hinf I digestion revealed a novel satellite, which we have called the C. limon satellite DNA 300 (CL300). Sequence analysis shows significant homology between a portion of the CL300 monomer and the transposase box of an En/Spm-like element. The CL300 satellite was also detected in grapefruit, sour orange, trifoliate orange and kumquat. These results suggest that the CL300 repeat is an ancient satellite, and we propose that a significant portion originated by amplification of a genomic region containing the En/Spm-like transposase element.     

The human GINS complex binds to and specifically stimulates human DNA polymerase alpha-primase

The eukaryotic GINS complex has an essential role in the initiation and elongation phases of genome duplication. It is composed of four paralogous subunits--Sld5, Psf1, Psf2 and Psf3--which are ubiquitous and evolutionarily conserved in eukaryotic organisms. Here, we report the biochemical characterization of the human GINS complex (hGINS). The four hGINS subunits were coexpressed in Escherichia coli in a highly soluble form and purified as a complex. hGINS was shown to interact directly with the heterodimeric human DNA primase, by using either surface plasmon resonance measurements or by immunoprecipitation experiments carried out with anti-hGINS antibodies. The DNA polymerase alpha-primase synthetic activity was specifically stimulated by hGINS on various primed DNA templates. The significance of these findings is discussed in view of the molecular dynamics at the human replication fork.     

Fibrin as a scaffold for cardiac tissue engineering

Fibrin is a natural biopolymer with many interesting properties, such as biocompatibility, bioresorbability, ease of processing, ability to be tailored to modify the conditions of polymerization, and potential for incorporation of both cells and cell mediators. Moreover, the fibrin network has a nanometric fibrous structure, mimicking extracellular matrix, and it can also be used in autologous applications. Therefore, fibrin has found many applications in tissue engineering, combined with cells, growth factors, or drugs. Because a major limitation of cardiac cell therapy is low cell engraftment, the use of biodegradable scaffolds for specific homing and in situ cell retention is desirable. Thus, fibrin-based injectable cardiac tissue engineering may enhance cell therapy efficacy. Fibrin-based biomaterials can also be used for engineering heart valves or cardiac patches. The aim of this review is to show cardiac bioengineering uses of fibrin, both as a cell delivery vehicle and as an implantable biomaterial.