Upregulation of oxidative stress markers in human microvascular endothelial cells by complexes of serum albumin and digestion products of glycated casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95°C for 5 h to give AGE‐casein (AGE‐Cas). Simulated stomach and small intestine digestion of AGE‐Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)‐LMM‐AGE‐Cas complexes. Stimulation of human microvascular endothelial cells with BSA‐LMM‐AGE‐Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin‐3 (AGE‐R3), tumor necrosis factor alpha, and a marker of the mitogen‐activated protein kinase pathway (MAPK‐1), as well as p65NF‐κB activation. Cells treated with LMM digestion products of AGE‐Cas significantly increased AGE‐R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA‐LMM‐AGE‐Cas and LMM‐AGE‐Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and downstream inflammatory pathways. AGE‐R3 may protect against these effects. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:364–372, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20301     

Initiation of embryogenic cell suspensions of taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> var. <Emphasis Type="Italic">esculenta</Emphasis>) and plant regeneration

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4-D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settled cell volume while approximately 60% of the embryos regenerated into plants.     

Nested areas of endemism analysis

Aim  To develop and evaluate a nested clade approach to aid in the determination of areas of endemism (AoE) in biogeographical studies.
Methods  We adapted the nested clade analysis (NCA) to studies of areas of endemism. For this purpose we adapted several of the programs currently in use. Two data sets were examined using this approach – one involving Sciobius in southern Africa and the other involving terrestrial mammals in Mexico.
Results  Nested clade analysis as applied to areas of endemism produced results similar to those of previous analyses of Sciobus in southern Africa. An analysis of terrestrial mammals in Mexico supports the designation of some biogeographical provinces as areas of endemism while suggesting that other provinces may comprise composite distributions that should be subdivided.
Main conclusions  The nested clade analysis approach utilized primarily in genetic analysis of phylogeographical patterns in population biology studies can be adapted to understanding AoE in the realm of biogeography. This approach offers a statistical paradigm to evaluate AoE suggested by parsimony analysis of endemicity (PAE) trees.     

Stability of pulsatile blood flow at the ostium of cerebral aneurysms

The strength and direction of blood flow into and within a cerebral aneurysm are important issues in developing effective interventional strategies to stabilize the aneurysm. We tested the hypothesis that there are significant major hemodynamic features that are common to many aneurysm flows of the type studied here. This was investigated by performing computational fluid dynamic simulations of flow near 7 cerebral aneurysms using geometrical data obtained from clinical CT scans. Our numerical simulations of flow across the ostium plane of an aneurysm show that in many cases there is relatively stable flow structure that is maintained over the phase of the pulsatile flow cycle. The two main features of this flow are (1) quasi-permanent regions of flow influx and efflux across the ostium plane exist, separated by a “virtual boundary”, and (2) a helical vortex flow pattern within the aneurismal sac with swirl in two orthogonal cross-sectional planes. These numerical observations are consistent with in vitro experimental data from ultrasound color-Doppler velocimetry and other numerical and experimental studies. The observed flow patterns are found to occur in different types of aneurysms (bifurcation and sidewall), and can persist even after flow parameters are perturbed beyond the normal range of physiological flow conditions. These results suggest that in many cases, major aspects of the behavior of aneurismal hemodynamics for important classes of aneurysms can be learned from an analysis of steady, non-pulsatile flow, which is simpler and faster to simulate than time-dependent, pulsatile flow. An understanding of this fluid dynamical behavior may also prove useful in the design of stents, coils, and various other endovascular flow diverting devices.     

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing

We report that combining a DNA analog (2′F-ANA) with rigid RNA analogs [2′F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1–1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.     

Short spacing between the Shine-Dalgarno sequence and P codon destabilizes codon-anticodon pairing in the P site to promote +1 programmed frameshifting

Myxococcus xanthus is a Gram‐negative bacterium capable of complex developmental processes involving vegetative swarming and fruiting body formation. Social (S‐) gliding motility, one of the two motility systems used by M. xanthus, requires at least two cell surface structures: type IV pili (TFP) and extracellular polysaccharides (EPS). Extended TFP that are composed of thousands of copies of PilA retract upon binding to EPS and thereby pull the cell forward. TFP also act as external sensor to regulate EPS production. In this study, we generated a random PilA mutant library and identified one derivative, SW1066, which completely failed to undergo developmental processes. Detailed characterization revealed that SW1066 produced very little EPS but wild‐type amounts of PilA. These mutated PilA subunits, however, are unable to assemble into functional TFP despite their ability to localize to the membrane. By preventing the mutated PilA of SW1066 to translocate from the cytoplasm to the membrane, fruiting body formation and EPS production were restored to the levels observed in mutant strains lacking PilA. This apparent connection between PilA membrane accumulation and reduction in surface EPS implies that specific cellular PilA localization are required to maintain the EPS level necessary to sustain normal S‐motility in M. xanthus.     

Structural investigation of a viral ortholog of human NEIL2/3 DNA glycosylases

Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.     

Pathotype and Genetic Diversity amongst Indian Isolates of Xanthomonas oryzae pv. oryzae

A number of rice resistance genes, called Xa genes, have been identified that confer resistance against various strains of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. An understanding of pathotype diversity within the target pathogen population is required for identifying the Xa genes that are to be deployed for development of resistant rice cultivars. Among 1024 isolates of Xoo collected from 20 different states of India, 11 major pathotypes were distinguished based on their reaction towards ten Xa genes (Xa1, Xa3, Xa4, xa5, Xa7, xa8, Xa10, Xa11, xa13, Xa21). Isolates belonging to pathotype III showing incompatible interaction towards xa8, xa13 and Xa21 and compatible interaction towards the rest of Xa genes formed the most frequent (41%) and widely distributed pathotype. The vast majority of the assayed Xoo isolates were incompatible with one or more Xa genes. Exceptionally, the isolates of pathotype XI were virulent on all Xa genes, but have restricted distribution. Considering the individual R-genes, Xa21 appeared as the most broadly effective, conferring resistance against 88 % of the isolates, followed in decreasing order by xa13 (84 %), xa8 (64 %), xa5 (30 %), Xa7 (17 %) and Xa4 (14 %). Fifty isolates representing all the eleven pathotypes were analyzed by southern hybridization to determine their genetic relatedness using the IS1112 repeat element of Xoo. Isolates belonging to pathotype XI were the most divergent. The results suggest that one RFLP haplotype that is widely distributed all over India and is represented in strains from five different pathotypes might be an ancestral haplotype. A rice line with xa5, xa13 and Xa21 resistance genes is resistant to all strains, including those belonging to pathotype XI. This three gene combination appears to be the most suitable Xa gene combination to be deployed in Indian rice cultivars.