Effects of Petunia cytoplasmic male sterile (CMS) cytoplasm on the development of sterile and fertility-restored P. parodii anthers

The developmental defects causing cytoplasmic male sterility in Petunia parodii are described in isonuclear fertile, sterile, and fertility-restored plants using both light- and scanning electron microscopy. The aberrant development of the sporogenous tissue and tapetal layer caused by the cytoplasmic male sterile cytoplasm in both Petunia hybrida and P. parodii nuclear backgrounds is similar in onset and progression. The degeneration of the sporogenous tissue and tapetal layer of sterile anthers is first apparent late in meiosis and results in highly abnormal sterile sporogenous tissue by tetrad stage of fertile anthers. The stomium and endothecium do not show major developmental differences between fertile and sterile anthers, but the inner connective tissue of sterile anthers contained calcium crystals not found at high abundance in fertile anthers. Ovoid bodies containing magnesium and phosphorus were seen only in the vascular bundles of fertile anthers. Material prepared for the scanning electron microscope by freeze drying showed better retention of fragile morphological features, while critical-point drying permitted examination of nonvolatile structures, such as cell walls.     

Identification and distribution of profilin in tomato (Lycopersicon esculentum Mill.)

Profilin is a G-actin monomer-binding protein which has been shown to participate in actin-based tipgrowth of animal cells. The abundance of profilin in pollen and its occurrence in several vegetable foods raises the question of the role of profilin in plants. First, its distribution throughout various parts of the plant needs to be determined. This paper describes observations on the presence of profilin in the tomato plant (Lycopersicon esculentum Mill.). The distribution of profilin in flower buds, stems, leaves, roots, and fruits of tomato was determined by immunoblotting and by tissue printing, showing that profilin is present in most if not all parts of the tomato plant.We gratefully acknowledge the help provided by Dr. A.T. Jagendorf and the donation of tomato seeds and maize pollen by N. Eanetta and Dr. M. Smith, respectively. The use of Dr. R. Wayne's SZH ILLD dissecting microscope is gratefully acknowledged. This work was aided by helpful discussions with C.S. Combs, Dr. C.A. Conley, and Dr. J. Andersland. This work was supported by a Hatch grant and NRI Competitive Grants Program/USDA 94-37304-1046 to MVP. This material is based upon work supported under a National Science Foundation Graduate Research Fellowship to DWD.     

Analysis of Species-Selectivity of Human,Mouse and Rat Cytochrome P450 1A and 2B Subfamily Enzymes using Molecular Modeling,Docking and Dynamics Simulations

Cytochrome P450 (CYP) 1A and 2B subfamily enzymes are important drug metabolizing enzymes, and are highly conserved across species in terms of sequence homology. However, there are major to minor structural and macromolecular differences which provide for species-selectivity and substrate-selectivity. Therefore, species-selectivity of CYP1A and CYP2B subfamily proteins across human, mouse and rat was analyzed using molecular modeling, docking and dynamics simulations when the chiral molecules quinine and quinidine were used as ligands. The three-dimensional structures of 17 proteins belonging to CYP1A and CYP2B subfamilies of mouse and rat were predicted by adopting homology modeling using the available structures of human CYP1A and CYP2B proteins as templates. Molecular docking and dynamics simulations of quinine and quinidine with CYP1A subfamily proteins revealed the existence of species-selectivity across the three species. On the other hand, in the case of CYP2B subfamily proteins, no role for chirality of quinine and quinidine in forming complexes with CYP2B subfamily proteins of the three species was indicated. Our findings reveal the roles of active site amino acid residues of CYP1A and CYP2B subfamily proteins and provide insights into species-selectivity of these enzymes across human, mouse, and rat.     

Discovery of chiral dihydropyridopyrimidinones as potent,selective and orally bioavailable inhibitors of AKT

During the course of our research efforts to develop potent and selective AKT inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones (DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against ROCK₂. A key challenge in this program was the poor physicochemical properties of the initial lead compound 5. Integration of structure-based drug design and physical properties-based design resulted in replacement of a highly hydrophobic poly fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound 6 with much improved physicochemical properties. Subsequent SAR studies led to the synthesis of new pyran analog 7 with improved cell potency. Further optimization of pharmacokintetics properties by increasing permeability with appropriate fluorinated alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the phosphorylation of GSK3β in vivo and had robust, dose and concentration dependent efficacy in the U87MG tumor xenograft model.     

Host Gut Motility Promotes Competitive Exclusion within a Model Intestinal Microbiota

The gut microbiota is a complex consortium of microorganisms with the ability to influence important aspects of host health and development. Harnessing this “microbial organ” for biomedical applications requires clarifying the degree to which host and bacterial factors act alone or in combination to govern the stability of specific lineages. To address this issue, we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within a vertebrate gut. We observed that the interplay between each population and the gut environment produces distinct spatiotemporal patterns. As a consequence, one species dominates while the other experiences sudden drops in abundance that are well fit by a stochastic mathematical model. Modeling revealed that direct bacterial competition could only partially explain the observed phenomena, suggesting that a host factor is also important in shaping the community. We hypothesized the host determinant to be gut motility, and tested this mechanism by measuring colonization in hosts with enteric nervous system dysfunction due to a mutation in the ret locus, which in humans is associated with the intestinal motility disorder known as Hirschsprung disease. In mutant hosts we found reduced gut motility and, confirming our hypothesis, robust coexistence of both bacterial species. This study provides evidence that host-mediated spatial structuring and stochastic perturbation of communities can drive bacterial population dynamics within the gut, and it reveals a new facet of the intestinal host–microbe interface by demonstrating the capacity of the enteric nervous system to influence the microbiota. Ultimately, these findings suggest that therapeutic strategies targeting the intestinal ecosystem should consider the dynamic physical nature of the gut environment.     

Synthesis and structure-activity relationships of novel pyrimido[1,2-b]indazoles as potential anticancer agents against A-549 cell lines

A series of novel pyrimido[1,2-b]indazoles 5, 7 have been prepared from 3-trifluoromethyl-5-phenyl-2,6-dicyano anilines 1 via novel indazole regioisomers 3 and 4 through a facile strategy. Specific examples were evaluated for anticancer activity in vitro and found to exhibit promising activity against A-549 cell lines and are more effective than Etoposide. QSAR models were developed and validated by cross-validation method. The results of the best QSAR model were further compared with the crystal structure of tubulin protein. The binding energies estimated were found to have a good correlation with the experimental inhibitory potencies.     

Nocturnal male sex drive in Drosophila

Many behaviors and physiological processes including locomotor activity, feeding, sleep, mating, and migration are dependent on daily or seasonally reoccurring, external stimuli. In D. melanogaster, one of the best-studied circadian behaviors is locomotion. The fruit fly is considered a diurnal (day active/night inactive) insect, based on locomotor-activity recordings of single, socially naive flies. We developed a new circadian paradigm that can simultaneously monitor two flies in simple social contexts. We find that heterosexual couples exhibit a drastically different locomotor-activity pattern than individual males, females, or homosexual couples. Specifically, male-female couples exhibit a brief rest phase around dusk but are highly active throughout the night and early morning. This distinct locomotor-activity rhythm is dependent on the clock genes and synchronized with close-proximity encounters, which reflect courtship, between the male and female. The close-proximity rhythm is dependent on the male and not the female and requires circadian oscillators in the brain and the antenna. Taken together, our data show that constant exposure to stimuli emanating from the female and received by the male olfactory and other sensory systems is responsible for the significant shift in intrinsic locomotor output of socially interacting flies.