Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Inhibition of mutation and combating the evolution of antibiotic resistance

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.     

CCL5/CCR5 axis promotes the motility of human oral cancer cells

CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 2009. © 2009 Wiley‐Liss, Inc.     

Uncialamycin as a novel payload for antibody drug conjugate (ADC) based targeted cancer therapy

Uncialamycin analogs were evaluated as potential cytotoxic agents in an antibody-drug conjugate (ADC) approach to treating human cancer. These analogs were synthesized using Hauser annulations of substituted phthalides as a key step. A highly potent uncialamycin analog 3c with a valine-citrulline dipeptide linker was conjugated to an anti-mesothelin monoclonal antibody (mAb) through lysines to generate a meso-13 conjugate. This conjugate demonstrated subnanomolar potency (IC50?=?0.88?nM, H226 cell line) in in vitro cytotoxicity experiments with good immunological specificity to mesothelin-positive lung cancer cell lines. The potency and mechanism of action of this uncialamycin class of enediyne antitumor antibiotics make them attractive payloads in ADC-based cancer therapy.     

High Conformational Variability in the GluK2 Kainate Receptor Ligand-Binding Domain

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Achieving Molecular Selectivity in Imaging Using Multiphoton Raman Spectroscopy Techniques

In the case of most optical imaging methods, contrast is generated either by physical properties of the sample (Differential Image Contrast, Phase Contrast), or by fluorescent labels that are localized to a particular protein or organelle. Standard Raman and infrared methods for obtaining images are based upon the intrinsic vibrational properties of molecules, and thus obviate the need for attached fluorophores. Unfortunately, they have significant limitations for live-cell imaging. However, an active Raman method, called Coherent Anti-Stokes Raman Scattering (CARS), is well suited for microscopy, and provides a new means for imaging specific molecules. Vibrational imaging techniques, such as CARS, avoid problems associated with photobleaching and photo-induced toxicity often associated with the use of fluorescent labels with live cells. Because the laser configuration needed to implement CARS technology is similar to that used in other multiphoton microscopy methods, such as two-photon fluorescence and harmonic generation, it is possible to combine imaging modalities, thus generating simultaneous CARS and fluorescence images. A particularly powerful aspect of CARS microscopy is its ability to selectively image deuterated compounds, thus allowing the visualization of molecules, such as lipids, that are chemically indistinguishable from the native species.     

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK‐dependent Cyclooxygenase‐2

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47^phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG₂ receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.     

Distinct roles of synapsin I and synapsin II during neuronal development.

The synapsins are a family of neuron-specific proteins, associated with the cytoplasmic surface of synaptic vesicles, which have been shown to regulate neurotransmitter release in mature synapses and to accelerate development of the nervous system. Using neuronal cultures from mice lacking synapsin I, synapsin II, or both synapsins I and II, we have now found that synapsin I and synapsin II play distinct roles in neuronal development. Deletion of synapsin II, but not synapsin I, greatly retarded axon formation. Conversely, deletion of synapsin I, but not synapsin II, greatly retarded synapse formation. Remarkably, the deletion of both synapsins led to partial restoration of the wild phenotype. The results suggest that the synapsins play separate but coordinated developmental roles.     

Enzymatic Characterization of Insecticide Resistance Mechanisms in Field Populations of Malaysian Culex quinquefasciatus Say (Diptera: Culicidae)

Background

There has been no comprehensive study on biochemical characterization of insecticide resistance mechanisms in field populations of Malaysian Culex quinquefasciatus. To fill this void in the literature, a nationwide investigation was performed to quantify the enzyme activities, thereby attempting to characterize the potential resistance mechanisms in Cx. quinquefasciatus in residential areas in Malaysia.

Methodology/Principal Findings

Culex quinquefasciatus from 14 residential areas across 13 states and one federal territory were subjected to esterases, mixed function oxidases, glutathione-S-transferase and insensitive acetylcholinesterase assays. Enzyme assays revealed that α-esterases and β-esterases were elevated in 13 populations and 12 populations, respectively. Nine populations demonstrated elevated levels of mixed function oxidases and glutathione-S-transferase. Acetylcholinesterase was insensitive to propoxur in all 14 populations. Activity of α-esterases associated with malathion resistance was found in the present study. In addition, an association between the activity of α-esterases and β-esterases was also demonstrated.

Conclusions/Significance

The present study has characterized the potential biochemical mechanisms in contributing towards insecticide resistance in Cx. quinquefasciatus field populations in Malaysia. Identification of mechanisms underlying the insecticide resistance will be beneficial in developing effective mosquito control programs in Malaysia.