Normalized transillumination of fluorescent proteins in small animals

We investigated the capacity of normalized transillumination methods in imaging fluorescent proteins in visible light, in phantoms and in live mice. We demonstrate significant imaging improvements over epi-illumination imaging, as commonly applied today. Of particular importance is the significant betterment in contrast achieved, due to minimization of the influence of autofluorescence and the enhancement of depth sensitivity compared to epi-illumination imaging. Although normalized transillumination does not match the accuracy and quantification capacity of tomographic imaging, it nevertheless attains key advantages over other planar optical imaging methods while preserving implementation simplicity. Due to the balance achieved between simplicity and accuracy, normalized transillumination approaches could serve as an important alternative molecular imaging method.     

Investigation of an Anomalously Accelerating Substitution in the Folding of a Prototypical Two-State Protein

The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.     

Chronic hyperglycemia predisposes to exaggerated inflammatory response and leukocyte dysfunction in Akita mice

The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1alpha, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.     

Natural Synchronisation for the Study of Cell Division in the Green Unicellular Alga Ostreococcus tauri

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.     

Therapeutic use of recombinant methionyl human leptin

Recombinant methionyl human leptin (r-metHuLeptin) was first used as a replacement therapy in patients bearing inactivating mutations in the leptin gene. In this indication, it was shown since 1999 to be very efficient in inducing a dramatic weight loss in rare children and adults with severe obesity due to the lack of leptin. These first clinical trials clearly showed that r-metHuLeptin acted centrally to reduce food intake, inducing loss of fat mass, and to correct metabolic alterations, immune and neuroendocrine defects. A few years later, r-metHuLeptin was also shown to reverse the metabolic complications associated with lipodystrophic syndromes, due to primary defects in fat storage, which induce leptin deficiency. The beneficial effects, which could be mediated by central and/or peripheral mechanisms, are thought to mainly involve the lowering effects of leptin on ectopic lipid storage, in particular in liver and muscles, reducing insulin resistance. Interestingly, r-metHuLeptin therapy also reversed the hypothalamic-pituitary-gonadal axis dysfunctions associated with hypothalamic amenorrhea. However, if r-metHuLeptin treatment has been shown to be dramatically efficient in leptin-deficient states, its very limited effect in inducing weight loss in common obese patients revealed that, in patients with adequate leptin secretion, mechanisms of leptin resistance and leptin tolerance prevent r-metHuLeptin from inducing any additional effects. This review will present the current data about the effects of r-metHuLeptin therapy in humans, and discuss the recent perspectives of this therapy in new indications.     

Pomegranate seed oil reduces intestinal damage in a rat model of necrotizing enterocolitis

Pomegranate seed oil (PSO), which is the major source of conjugated linolenic acids such as punicic acid (PuA), exhibits strong anti-inflammatory properties. Necrotizing enterocolitis (NEC) is a devastating disease associated with severe and excessive intestinal inflammation. The aim of this study was to evaluate the effects of orally administered PSO on the development of NEC, intestinal epithelial proliferation, and cytokine regulation in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), formula-fed rats (FF), or rats fed with formula supplemented with 1.5% of PSO (FF + PSO). All groups were exposed to asphyxia/cold stress to induce NEC. Intestinal injury, epithelial cell proliferation, cytokine production, and trefoil factor 3 (Tff3) production were evaluated in the terminal ileum. Oral administration of PSO (FF+PSO) decreased the incidence of NEC from 61 to 26%. Feeding formula with PSO improved enterocyte proliferation in the site of injury. Increased levels of proinflammatory IL-6, IL-8, IL-12, IL-23, and TNF-α in the ileum of FF rats were normalized in PSO-treated animals. Tff3 production in the FF rats was reduced compared with DF but not further affected by the PSO. In conclusion, administration of PSO protects against NEC in the neonatal rat model. This protective effect is associated with an improvement of intestinal epithelial homeostasis and a strong anti-inflammatory effect of PSO on the developing intestinal mucosa.