Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals

Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.     

Renal clearance of quantum dots

The field of nanotechnology holds great promise for the diagnosis and treatment of human disease. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or their biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. Using intravenously administered quantum dots in rodents as a model system, we have precisely defined the requirements for renal filtration and urinary excretion of inorganic, metal-containing nanoparticles. Zwitterionic or neutral organic coatings prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter by >15 nm and prevented renal excretion. A final hydrodynamic diameter <5.5 nm resulted in rapid and efficient urinary excretion and elimination of quantum dots from the body. This study provides a foundation for the design and development of biologically targeted nanoparticles for biomedical applications.     

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.     

Generation of a naïve/synthetic antibody specific to botulinum neurotoxin via motif-grafting

In this study, we describe a new approach to the production of naïve/synthetic human antibodies against the botulinum neurotoxin (BoNT). First, peptides that bind to BoNT serotype A (BoNT/A) were screened from a phage display of a combinatorial peptide library. One peptide, designated ANT 12-2 (TLPSPLALLTVH), was determined to interact with BoNT/A, as well as with other serotypes of BoNT. This peptide specifically reacted with the native form of BoNT/A, but not with its formalin-inactivated form. Next, a hybrid naïve/synthetic human Fab library was generated via the grafting of a peptide motif from ANT 12-2 into HCDR3 with randomized flanking residues. Through biopanning, the Fab clone, ANTHU-1, which harbors the HCDR3 sequence of VRIQRSPLALLSWGDV, was selected and confirmed in order to retain the same BoNT-binding characteristics as ANT 12-2.     

Mouse strain-dependent osteoclastogenesis in response to lipopolysaccharide

Bacterial lipopolysaccharide (LPS) is a potent stimulator of bone resorption in periodontitis. Co-culture systems of mouse calvaria-derived osteoblasts and bone marrow-derived preosteoclasts were used as an in vitro osteoclast differentiation. This study revealed that co-cultures using ddY or ICR mouse strain responded differently to LPS while responded equally to 1alpha,25(OH)2D3. Thus, the different response to LPS indicates dissimilarity of two mouse stains in their capacity for generating osteoclasts while the two mouse strains share the similarity in response to 1alpha,25(OH)2D3. To identify which cells between osteoblasts and preosteoclasts in the co-culture are responsible for the dissimilarity, the reciprocal co-cultures were performed between ddY and ICR mouse strains. The treatment of 1,25(OH)2D3 to ddY/ICR (osteoblasts from ddY/preosteoclasts from ICR) and ICR/ddY reciprocal co-cultures also showed the similarity. In case of LPS treatment, the results of ddY/ICR were similar to ddY/ddY and the results of the other reciprocal co-culture, ICR/ddY combination, were consistent with those of ICR/ICR. It suggests that the dissimilarity between the two mouse strains may resident in osteoblasts but not in preosteoclasts. Therefore, the osteoblast is responsible for mouse strain-dependent osteoclastogenesis in response to LPS. Although mouse models will continue to provide insights into molecular mechanisms of osteoclastogenesis, caution should be exercised when using different mouse strains, especially ddY and ICR strains as models for osteoclast differentiation.     

Modeling of poly(3-hydroxybutyrate) production by high cell density fed-batch culture of R<Emphasis Type="Italic">alstonia eutropha</Emphasis>

High cell density culturing has been conducted for the production of poly(3-hydroxybutyrate) fed-batch cultures ofRalstonia eutropha with phosphate limitation. It was found that a high glucose concentration inhibited the synthesis of P(3HB) in the high cell density culture ofR. eutropha. Although a low glucose concentration can trigger the synthesis of P(3HB) in a manner similar to that of phosphate limitation, it also limited both the P(3HB) synthesis and the cell growth, and led to a low P(3HB) productivity because glucose is the sole carbon source in this reaction. An unstructured model was proposed for predicting the cell growth and P(3HB) synthesis in high cell density cultures ofR. eutropha, where the phosphate concentration played a key role in the accumulation of P(3HB) and in cell growth. Good agreements were found between the experimental data and model predictions. The results of simulation showed that the final P(3HB) concentration would decrease more than 25% when the glucose was concentration increased to 40 g/L, and indicated that the optimal glucose concentration for P(3HB) production by high cell density cultures ofR. eutropha was around 9 g/L.     

Enhancement of erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate addition

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.     

Identification and functional analysis of vibrio vulnificus SmcR, a novel global regulator

Recently, quorum sensing has been implicated as an important global regulator controlling the production of numerous virulence factors such as capsular polysaccharides in bacterial pathogens. The nucleotide and deduced amino acid sequences of smcR, a homolog of V. harveyi luxR identified from V. vulnificus ATCC29307, were analyzed. The amino acid sequence of SmcR from V. vulnificus was 72 to 92% similar to those of LuxR homologs from Vibrio spp. Functions of SmcR were assessed by the construction of an isogenic mutant, whose smcR gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of smcR resulted in a significant alteration in biofilm formation, in type of colony morphology, and in motility. When compared with the wild-type, the smcR mutant exhibited reduced survival under adverse conditions, such as acidic pH and hyperosmotic stress. The smcR mutant exhibited decreased cytotoxic activity toward INT 407 cells in vitro. Furthermore, the intraperitoneal LD50 of the smcR mutant was approximately 10(2) times higher than that of parental wild-type. Therefore, it appears that SmcR is a novel global regulator, controlling numerous genes contributing to the pathogenesis as well as survival of V. vulnificus.