Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

The mouse arylamine N-acetyltransferase 2 (Nat2) and its homologue (NAT1) in humans are known to detoxify xenobiotic arylamines and are also thought to play a role in endogenous metabolism. Human NAT1 is highly over-expressed in estrogen receptor positive breast tumours and is implicated in susceptibility to neural tube defects. In vitro assays have suggested an endogenous role for human NAT1 in folate metabolism, but in vivo evidence to support this hypothesis has been lacking. Mouse Nat2 provides a good model to study human NAT1 as it shows similar expression profiles and substrate specificities. We have generated transgenic mice lacking a functional Nat2 gene and compared the urinary levels of acetylated folate metabolite para-aminobenzoylglutamate in Nat2 knockout and Nat2 wild-type mice. These results support an in vivo role for mouse Nat2/human NAT1 in folate metabolism. In addition, effects of the Nat2 deletion on sex ratios and neural tube development are described.     

Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism

Lon is a homo-oligomeric ATP-dependent serine protease that functions in the degradation of damaged and certain regulatory proteins. This enzyme has emerged as a novel target in the development of antibiotics because of its importance in conferring bacterial virulence. In this study, we explored the mechanism by which the proteasome inhibitor MG262, a peptidyl boronate, inhibits the peptide hydrolysis activity of Salmonella enterica serovar Typhimurium Lon. In addition, we synthesized a fluorescent peptidyl boronate inhibitor based upon the amino acid sequence of a product of peptide hydrolysis by the enzyme. Using steady-state kinetic techniques, we have shown that two peptidyl boronate variants are competitive inhibitors of the peptide hydrolysis activity of Lon and follow the same two-step, time-dependent inhibition mechanism. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. This is followed by a second slow step in which Lon undergoes a conformational change or isomerization to increase the interaction of the inhibitor with the proteolytic active site to yield an overall inhibition constant of 5-20 nM. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease that has required the binding of ATP in order to observe inhibition.     

Synthesis and biophysical evaluation of minor-groove binding C-terminus modified pyrrole and imidazole triamide analogs of distamycin

Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.     

Simulation of human atherosclerotic femoral plaque tissue: the influence of plaque material model on numerical results

Background

Due to the limited number of experimental studies that mechanically characterise human atherosclerotic plaque tissue from the femoral arteries, a recent trend has emerged in current literature whereby one set of material data based on aortic plaque tissue is employed to numerically represent diseased femoral artery tissue. This study aims to generate novel vessel-appropriate material models for femoral plaque tissue and assess the influence of using material models based on experimental data generated from aortic plaque testing to represent diseased femoral arterial tissue.

Methods

Novel material models based on experimental data generated from testing of atherosclerotic femoral artery tissue are developed and a computational analysis of the revascularisation of a quarter model idealised diseased femoral artery from a 90% diameter stenosis to a 10% diameter stenosis is performed using these novel material models. The simulation is also performed using material models based on experimental data obtained from aortic plaque testing in order to examine the effect of employing vessel appropriate material models versus those currently employed in literature to represent femoral plaque tissue.

Results

Simulations that employ material models based on atherosclerotic aortic tissue exhibit much higher maximum principal stresses within the plaque than simulations that employ material models based on atherosclerotic femoral tissue. Specifically, employing a material model based on calcified aortic tissue, instead of one based on heavily calcified femoral tissue, to represent diseased femoral arterial vessels results in a 487 fold increase in maximum principal stress within the plaque at a depth of 0.8 mm from the lumen.

Conclusions

Large differences are induced on numerical results as a consequence of employing material models based on aortic plaque, in place of material models based on femoral plaque, to represent a diseased femoral vessel. Due to these large discrepancies, future studies should seek to employ vessel-appropriate material models to simulate the response of diseased femoral tissue in order to obtain the most accurate numerical results.

     

Integration of RNA processing and expression level control modulates the function of the Drosophila Hox gene Ultrabithorax during adult development

Although most metazoan genes undergo alternative splicing, the functional relevance of the majority of alternative splicing products is still unknown. Here we explore this problem in the Drosophila Hox gene Ultrabithorax (Ubx). Ubx produces a family of six protein isoforms through alternative splicing. To investigate the functional specificity of the Ubx isoforms, we studied their role during the formation of the Drosophila halteres, small dorsal appendages that are essential for normal flight. Our work shows that isoform Ia, which is encoded by all Ubx exons, is more efficient than isoform IVa, which lacks the amino acids coded by two small exons, in controlling haltere development and regulating Ubx downstream targets. However, our experiments also demonstrate that the functional differences among the Ubx isoforms can be compensated for by increasing the expression levels of the less efficient form. The analysis of the DNA-binding profiles of Ubx isoforms to a natural Ubx target, spalt, shows no major differences in isoform DNA-binding activities, suggesting that alternative splicing might primarily affect the regulatory capacity of the isoforms rather than their DNA-binding patterns. Our results suggest that to obtain distinct functional outputs during normal development genes must integrate the generation of qualitative differences by alternative splicing to quantitative processes affecting isoform protein expression levels.     

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.     

Investigation of cellular and molecular responses to pulsed focused ultrasound in a mouse model

Continuous focused ultrasound (cFUS) has been widely used for thermal ablation of tissues, relying on continuous exposures to generate temperatures necessary to induce coagulative necrosis. Pulsed FUS (pFUS) employs non-continuous exposures that lower the rate of energy deposition and allow cooling to occur between pulses, thereby minimizing thermal effects and emphasizing effects created by non-thermal mechanisms of FUS (i.e., acoustic radiation forces and acoustic cavitation). pFUS has shown promise for a variety of applications including drug and nanoparticle delivery; however, little is understood about the effects these exposures have on tissue, especially with regard to cellular pro-homing factors (growth factors, cytokines, and cell adhesion molecules). We examined changes in murine hamstring muscle following pFUS or cFUS and demonstrate that pFUS, unlike cFUS, has little effect on the histological integrity of muscle and does not induce cell death. Infiltration of macrophages was observed 3 and 8 days following pFUS or cFUS exposures. pFUS increased expression of several cytokines (e.g., IL-1α, IL-1β, TNFα, INFγ, MIP-1α, MCP-1, and GMCSF) creating a local cytokine gradient on days 0 and 1 post-pFUS that returns to baseline levels by day 3 post-pFUS. pFUS exposures induced upregulation of other signaling molecules (e.g., VEGF, FGF, PlGF, HGF, and SDF-1α) and cell adhesion molecules (e.g., ICAM-1 and VCAM-1) on muscle vasculature. The observed molecular changes in muscle following pFUS may be utilized to target cellular therapies by increasing homing to areas of pathology.     

Microbial community composition in sediments resists perturbation by nutrient enrichment

Functional redundancy in bacterial communities is expected to allow microbial assemblages to survive perturbation by allowing continuity in function despite compositional changes in communities. Recent evidence suggests, however, that microbial communities change both composition and function as a result of disturbance. We present evidence for a third response: resistance. We examined microbial community response to perturbation caused by nutrient enrichment in salt marsh sediments using deep pyrosequencing of 16S rRNA and functional gene microarrays targeting the nirS gene. Composition of the microbial community, as demonstrated by both genes, was unaffected by significant variations in external nutrient supply in our sampling locations, despite demonstrable and diverse nutrient-induced changes in many aspects of marsh ecology. The lack of response to external forcing demonstrates a remarkable uncoupling between microbial composition and ecosystem-level biogeochemical processes and suggests that sediment microbial communities are able to resist some forms of perturbation.     

Upstream stimulatory factor 1 associated with familial combined hyperlipidemia, LDL cholesterol, and triglycerides

Positive evidence has been reported for linkage and association between the upstream stimulatory factor 1 gene (USF1) and familial combined hyperlipidemia (FCHL). We genotyped the two most positive single-nucleotide polymorphisms (SNPs) (usf1s1: rs3737787 and usf1s2: rs2073658) from previous studies in a large family sample. This sample included 2,195 subjects in 87 Utah pedigrees ascertained for early death due to coronary heart disease (CHD), early strokes, or early onset hypertension. There were a total of 262 relative pairs in these families with FCHL. In the full family sample, FCHL was associated with usf1s1 (P=0.02). Triglyceride and LDL cholesterol defined qualitatively or quantitatively were also associated with usf1s1 (P=0.02–0.05). Results were strengthened for qualitative and quantitative triglyceride and LDL cholesterol when data from males only was analyzed, revealing associations for usf1s1 (P=0.001–0.02), usf1s2 (P=0.02–0.05) and the haplotype of these two SNPs (P=0.01–0.04). The strongest results were in the subset of subjects from families ascertained for premature stroke or hypertension, rather than those ascertained for premature CHD. This study replicates the involvement of USF1 in FCHL and related lipid traits in a family sample not ascertained for FCHL.