Neuropeptide orphanin FQ inhibits dendritic morphogenesis through activation of RhoA

Brain‐derived neurotrophic factor (BDNF) plays a facilitatory role in neuronal development and promotion of differentiation. Mechanisms that oppose BDNF's stimulatory effects create balance and regulate dendritic growth. However, these mechanisms have not been studied. We have focused our studies on the BDNF‐induced neuropeptide OrphaninFQ/ Nociceptin (OFQ); while BDNF is known to enhance synaptic activity, OFQ has opposite effects on activity, learning, and memory. We have now examined whether OFQ provides a balance to the stimulatory effects of BDNF on neuronal differentiation in the hippocampus. Golgi staining in OFQ knockout (KO) mice revealed an increase in primary dendrite length as well as spine density, suggesting that endogenous OFQ inhibits dendritic morphology. We have also used cultured hippocampal neurons to demonstrate that exogenous OFQ has an inhibitory effect on dendritic growth and that the neuropeptide alters the response to BDNF when pre‐administered. To determine if BDNF and OFQ act in a feedback loop, we inhibited the actions of the BDNF and OFQ receptors, TrkB and NOP using ANA‐12 and NOP KO mice respectively but our data suggest that the two factors do not act in a negative feedback loop. We found that the inhibition of dendritic morphology induced by OFQ is via enhanced RhoA activity. Finally, we have evidence that RhoA activation is required for the inhibitory effects of OFQ on dendritic morphology. Our results reveal basic mechanisms by which neurons not only regulate the formation of proper dendritic growth during development but also control plasticity in the mature nervous system. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 769–784, 2013     

Synthesis, characterization, X-ray structure and DNA photocleavage by cis-dichloro bis(diimine) Co(III) complexes

Complexes of the type [Co(LL)2Cl2]Cl, where LL = N,N'-ethylenediamine (en), 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (phendione) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) have been synthesized and characterized by elemental analyses, IR, UV-visible and NMR spectroscopy. Crystal structure of [Co(phendione)2Cl2]Cl x 0.5 HCl x 3.5 H2O has been solved and refined to R = 0.0552. The crystal is monoclinic with space group C2/c; a = 25.730(2) A, b = 12.375(1) A, c = 18.979(2) A, beta = 119.925(1) degrees and Z = 8. The DNA binding characteristics of the complexes, investigated by covalent binding assay, viscosity measurements and competitive binding fluorescence measurements show that the complexes interact with DNA covalently except the complex containing the planar dppz ligand which intercalates within the base pairs of DNA. The complexes containing en, phen and phendione cleave plasmid pBR 322 DNA upon irradiation under aerobic conditions while the complex containing the dppz ligand cleaves DNA upon irradiation under inert atmosphere. Molecular modeling studies show that the minimized structure of [Co(phendione)2Cl2]+, maintained the octahedral structure while binding to the N7 of guanines and the ligand fits into the major groove without disrupting the helical structure of the B-DNA.     

Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria

Mitochondria are tailored to meet the metabolic and signaling needs of each cell. To explore its molecular composition, we performed a proteomic survey of mitochondria from mouse brain, heart, kidney, and liver and combined the results with existing gene annotations to produce a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle. The protein expression data were largely concordant with large-scale surveys of RNA abundance and both measures indicate tissue-specific differences in organelle composition. RNA expression profiles across tissues revealed networks of mitochondrial genes that share functional and regulatory mechanisms. We also determined a larger "neighborhood" of genes whose expression is closely correlated to the mitochondrial genes. The combined analysis identifies specific genes of biological interest, such as candidates for mtDNA repair enzymes, offers new insights into the biogenesis and ancestry of mammalian mitochondria, and provides a framework for understanding the organelle's contribution to human disease.     

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.     

The solution NMR structure of Antheraea polyphemus PBP provides new insight into pheromone recognition by pheromone-binding proteins

Pheromone-binding proteins (PBPs) located in the antennae of male moth species play an important role in olfaction. They are carrier proteins, believed to transport volatile hydrophobic pheromone molecules across the aqueous sensillar lymph to the membrane-bound G protein-coupled olfactory receptor proteins. The roles of PBPs in molecular recognition and the mechanisms of pheromone binding and release are poorly understood. Here, we report the NMR structure of a PBP from the giant silk moth Antheraea polyphemus. This is the first structure of a PBP with specific acetate-binding function in vivo. The protein consists of nine alpha-helices: alpha1a (residues 2-5), alpha1b (8-12), alpha1c (16-23), alpha2 (27-34), alpha3a (46-52), alpha3b (54-59), alpha4 (70-79), alpha5 (84-100) and alpha6 (107-125), held together by three disulfide bridges: 19-54, 50-108 and 97-117. A large hydrophobic cavity is located inside the protein, lined with side-chains from all nine helices. The acetate-binding site is located at the narrow end of the cavity formed by the helices alpha3b and alpha4. The pheromone can enter this cavity through an opening between the helix alpha1a, the C-terminal end of the helix alpha6, and the loop between alpha2 and alpha3a. We suggest that Trp37 may play an important role in the initial interaction with the ligand. Our analysis also shows that Asn53 plays the key role in recognition of acetate pheromones specifically, while Phe12, Phe36, Trp37, Phe76, and Phe118 are responsible for non-specific binding, and Leu8 and Ser9 may play a role in ligand chain length recognition.     

Comparison of in vivo segmental foot motion during walking and step descent in patients with midfoot arthritis and matched asymptomatic control subjects

The purpose of this study was to compare in vivo segmental foot motion during walking and step descent in patients with midfoot arthritis and asymptomatic control subjects. Segmental foot motion during walking and step descent was assessed using a multi-segment foot model in 30 patients with midfoot arthritis and 20 age, gender and BMI matched controls. Peak and total range of motion (ROM), referenced to subtalar neutral, were examined for each of the following dependent variables: 1st metatarso-phalangeal (MTP1) dorsiflexion, 1st metatarsal (MT1) plantarflexion, ankle dorsiflexion, calcaneal eversion and forefoot abduction. The results showed that, compared to level walking, step descent required greater MTP1 dorsiflexion (p<0.01), MPT1 plantarflexion (p<0.01), ankle dorsiflexion (p<0.01), calcaneus eversion (p=0.03) and forefoot abduction (p=0.01), in all subjects. In addition, step descent also necessitated greater MTP1 dorsiflexion (p<0.01), ankle dorsiflexion (p<0.01) and forefoot abduction (p=0.02) excursion compared to walking. Patients with midfoot arthritis responded differently to the step task compared to control subjects in terms of MT1 and calcaneus eversion excursion. During walking, patients with midfoot arthritis showed significantly less MT1 plantarflexion excursion compared to control subjects (p=0.03). However, during step descent, both groups showed similar MT1 plantarflexion excursion. During walking, patients with midfoot arthritis showed similar calcaneus eversion excursion compared to control subjects. However, during step descent, patients with midfoot arthritis showed significantly greater calcaneus eversion excursion compared to control subjects (p=0.03). Independently or in combination, these motions may contribute to articular stress and consequently to symptoms in patients with midfoot arthritis.     

Immunotherapy of cancer with dendritic-cell-based vaccines

 Animal studies have shown that vaccination with genetically modified tumor cells or with dendritic cells (DC) pulsed with tumor antigens are potent strategies to elicit protective immunity in tumor-bearing animals, more potent than “conventional” strategies that have been tested in clinical settings with limited success. While both vaccination strategies are forms of cell therapy requiring complex and costly ex vivo manipulations of the patient’s cells, current protocols using dendritic cells are considerably simpler and would be more widely available. Vaccination with defined tumor antigens presented by DC has obvious appeal. However, in view of the expected emergence of antigen-loss variants as well as natural immunovariation, effective vaccine formulations must contain mixtures of commonly, if not universally, expressed tumor antigens. When, or even if, such common tumor antigens will be identified cannot be, predicted, however. Thus, for the foreseeable future, vaccination with total-tumor-derived material as source of tumor antigens may be preferable to using defined tumor antigens. Vaccination with undefined tumor-derived antigens will be limited, however, by the availability of sufficient tumor tissue for antigen preparation. Because the mRNA content of single cells can be amplified, tumor mRNA, or corresponding cDNA libraries, offer an unlimited source of tumor antigens. DC transfected with tumor RNA were shown to engender potent antitumor immunity in animal studies. Thus, immunotherapy using autologous DC loaded with unfractionated tumor-derived antigens in the form of RNA emerges as a potentially powerful and broadly useful vaccination strategy for cancer patients. Received: 10 October 1997 / Accepted: 12 January 1998     

Formulation of medium constituents by multiresponse analysis of central composite design to enhance chitinase production in Pantoea dispersa

In the present study, a high chitinase producing strain Pantoea dispersa was isolated from the sea dumps at Bhavnagar, India. Chitin, urea, CaCl2 and MgSO4 x 7H2O were variables used in central composite design for chitinase production. Chitinase, biomass and pH were the responses used in different models to evaluate individually fit ones. Quadratic model was found to be fit for chitinase response whereas in the case of biomass and pH, linear model was found to be fit without the effect of others. Chitinase production was optimized with respect to other responses such as biomass and pH in multiresponse analysis of response surface design by using desirability approach. In multiresponse analysis, following medium formulation (g/l), chitin, 15; urea, 0.32; CaCl2, 0.10 and MgSO4 x 7H2O, 0.08 was found to predict optimum chitinase production of 482.77 units/ml with overall highest desirability of 0.854 as compared to other formulations. The selection of model was done on the basis of high Adjusted R-squared value and lowered p-value for each model in individual analysis of each response. In multiresponse experiment, it was found that for response chitinase quadratic model and for responses pH and biomass linear models were well fit. Through desirability analysis, it was found that in the chitinase production, pH was essential as compared to biomass in P. dispersa. Endochitinase and chitobiase actvities were also studied.