Dissolution of Marriage - Fresh Air in Family Court

The state has a vital interest in marriage and divorce as a matter of public policy. Divorce is not simply a private controversy between husband and wife. The family relationship is the basis of our society, and its preservation is a matter of state concern. Publication or circulation of any article with the intent to procure or aid in procuring divorces, either in this state or elsewhere is made a crime punishable by a fine of $25 to$500 and/or six months\u27 imprisonment

Spatial intensity distribution analysis: studies of G Protein-coupled receptor oligomerization

Spatial intensity distribution analysis (SpIDA) is a recently developed approach for determining quaternary structure information on fluorophore-labelled proteins of interest in situ. It can be applied to live or fixed cells and native tissue. Using confocal images, SpIDA generates fluorescence intensity histograms that are analysed by super-Poissonian distribution functions to obtain density and quantal brightness values of the fluorophore-labelled protein of interest. This allows both expression level and oligomerisation state of the protein to be determined. We describe the application of SpIDA to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at steady state and following cellular challenge, and consider how SpIDA may be used to explore GPCR quaternary organisation in pathophysiology and to stratify medicines

Ligand regulation of the quaternary organization of cell surface M3 muscarinic acetylcholine receptors analyzed by fluorescence resonance energy transfer (FRET) imaging and homogenous time-resolved FRET

Flp-In T-REx 293 cells expressing a wild type human M muscarinic acetylcholine receptor construct constitutively and able to express a Receptor Activated Solely by Synthetic Ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine-N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET-donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogenous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M receptor expressed stably in Flp-In TREx 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used

Dynamic regulation of quaternary organization of the M1 muscarinic receptor by subtype-selective antagonist drugs

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands is unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by employing Spatial Intensity Distribution Analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules.microm-2 of the human muscarinic M1 receptor identified an ~75/25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter-term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked upregulation of the receptor, simple mass-action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior

Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor

Previous studies have indicated that the G protein-coupled secretin receptor is present as a homo-dimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated, is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and Spatial Intensity Distribution Analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed demonstration that the Epidermal Growth Factor receptor is predominantly monomeric in the absence of ligand and whilst wild type receptor was rapidly converted to a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that at moderate expression levels the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. By contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein-protein interactions between copies of the receptor polypeptide, whilst short term treatment with secretin had no effect on organization of the wild type receptor but increased the dimeric proportion of the mutated receptor variant

STRAIN-ENABLED PHOSPHINATION AND FLUORINATION REACTIONS

Chapter one of this thesis discusses the hydrophosphination of carbodiimides with secondary phosphine boranes. These reactions provide access to phosphaguanidine boranes, which have previously not been studied. A cyclic carbodiimide was also prepared, and hydrophosphination of this substrate provided access to structurally unique phosphaguanidines that bear resemblance to cyclophanes. Chapter two delineates the extension of this hydrophosphination method to functionalize strained carbocycles, namely bicyclo[1.1.0]butanes. The scope and stereoselectivity of this transformation was investigated, and further transformations of the cyclobutyl phosphine products were conducted. Studies on the addition of aromatic and heteroaromatic thiols to bicyclo[1.1.0]butanes are also described in this chapter. Chapter three describes a strain-relieving deoxyfluorination of bicyclo[1.1.0]butyl alcohols. The products of these transformations are fluorinated methylenecyclobutanes, which can be oxidatively transformed into cyclobutanones. Bicyclo[1.1.0]butyl amides were also subjected to fluorination with a high degree of diastereoselectivity. Chapter four details the development of bicyclo[1.1.0]butyl amides as potential androgen receptor antagonists for the treatment of castration-resistant prostate cancer. In the course of these medicinal chemistry studies, an acid-mediated isomerization of bicyclo[1.1.0]butanes to cyclobutenes was discovered

A molecular basis for selective antagonist destabilization of dopamine D3 receptor quaternary organization

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D3R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D3R dimerization

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community