The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M₁ muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M₁ and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M₁-mEGFP was expressed at levels equal to the M₁ receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M₁-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M₁-mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M₁-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Measuring and understanding the extent and potential significance of quaternary organization of members of the class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs) have both fascinated and frustrated researchers for many years (1, 2). Over time, a wide range of methods have been applied to address this question, and many different GPCRs have been examined. Outcomes have ranged from assertions that such receptors are monomeric and that results consistent with other conclusions reflect either artifacts of the method of measurement or that studies have been performed at nonphysiological levels of expression of the receptor being studied, to those that have suggested rather stable dimeric or tetrameric complexes (1). Only in the case of rhodopsin, the photon receptor expressed at very high levels (in the range of 24,000–30,000 molecules/µm²) in rod outer segments of the eye, have detailed studies been conducted in situ on a class A GPCR. In this example, various studies have shown that rhodopsin is organized as rows of dimers (3, 4). However, to our knowledge, no other GPCR is expressed natively at levels akin to rhodopsin. As such, although a substantial number of studies, generally performed in transfected cell lines or in artificial bilayer systems, have provided evidence that other GPCRs can and do form dimeric and/or higher-order quaternary complexes in a concentration-dependent manner (1, 2), how levels of expression required to observe such complexes relate to expression levels in native cells and tissues has been poorly defined, as is the stability of such complexes and whether they are regulated by ligand binding.Developments in fluorescence fluctuation analysis (FFA) have facilitated efforts to define the oligomeric status of transmembrane receptor proteins (5, 6). Unlike methods based on resonance energy transfer, only a single fluorophore-linked protein is required to be expressed to use FFA. It is, therefore, more practical to use such methods in native cells and tissues if linked to genome-editing approaches and/or the generation of transgenic “knock-in” animal models in which a receptor of interest is replaced with a fluorophore-tagged, modified form of the receptor. Moreover, the recent introduction of fluorescence intensity fluctuation (FIF) spectrometry (7–10) has overcome issues with other methods based on FFA that result in information being compressed due to averaging of oligomer-size data from interrogated regions of interest (RoIs) in which complex mixtures of oligomers of different sizes may be present (7, 8).To define whether the class A M₁ muscarinic acetylcholine receptor is present in hippocampal and cortical neurons as strict monomers or as a range of monomeric, dimeric, and, potentially, oligomeric complexes, we applied FIF spectrometry to images of such neurons isolated from a line of transgenic mice in which we replaced the M₁ receptor with a form of the receptor that includes C-terminally linked monomeric enhanced green fluorescent protein (mEGFP). We first show that both expression levels and function of the introduced M₁-mEGFP construct appear equivalent to the native M₁ receptor in wild-type (WT) mice, using a range of methods and measures ranging from [³H]ligand binding and cell signaling assays to locomotion. We then demonstrate in hippocampal and cortical neurons that in the basal state, the M₁-mEGFP construct is present as a mixture of monomers and dimeric or oligomeric complexes. We also show that the presence of either an agonist or an antagonist ligand promotes monomerization of the receptor. In these studies, we combined analysis of images of a fluorophore-modified receptor in situ with calculation of receptor oligomer complexity. The studies provide a clear and unambiguous answer to a long-standing question that has been the subject of considerable debate (11–13) but that has previously been restricted to studies performed on transfected cell lines. Moreover, these studies are a model for subsequent studies for researchers who plan to explore the topic of dimerization of rhodopsin-family GPCRs.     

Effect of Iontophoresis in Combination with Ionic Enhancers on the Lipid Structure of the Stratum Corneum: An X-ray Diffraction Study

Pharmaceutical Research -     

Bone Mineral Density in Chronic Kidney Disease Use and Misuse

Low bone mineral density (BMD) is a strong risk factor for low trauma fractures in the postmenopausal population without known chronic kidney disease (CKD). In stage 1?C3 CKD, low BMD can also be used to predict fracture risk with the gradient of risk similar to patients without CKD even though patients with stage 3 CKD have an approximate doubling of risk compared with age-matched patients without CKD. This greater risk of fracture in stage 3 CKD is not calculated in the current FRAX model. In stage 4?C5 CKD, BMD by dual-energy x-ray absorptiometry (DXA) is a poor predictor of fracture risk probably related to the severe derangements in bone metabolism in severe CKD, which alter bone quality and strength not measured by DXA. Serial BMD by DXA, however, may be useful in all stages of CKD to monitor for potential loss of BMD or effect of pharmacological agents to improve BMD. Newer radiological technologies, particularly high-resolution peripheral quantitative computerized tomography (HRpQcT) of the radius and tibia show promise to define the microstructural changes in bone that explain the greater risk of fracture observed in patients with CKD versus patients without CKD. BMD by DXA may still be of value across the spectrum of CKD, but physicians should realize its limitations and understand the greater risk of fracture in patients in all stages of CKD as compared to age-matched and BMD-matched patients without CKD.     

Leakage associated with intermediate restorative material and glass-ionomer cement retrograde fillings: a human and sheep teeth comparison with 2 different aging procedures.

OBJECTIVES: Leakage around retrograde fillings is an important cause of endodontic surgery. This in vitro study sought to compare the following: (1) methylene blue dye leakage linked to retrofillings in human and sheep teeth with the degree of dye penetration when intermediate restorative materials and Chemfil were used as retrofillings, (2) the apical microleakage in filled with that in unfilled root canals, and (3) 2 storage techniques, incubator-based and subcutaneous implantation in rats. STUDY DESIGN: Tested were 198 human and 196 sheep teeth that were retrofilled with intermediate restorative material or Chemfil, then stored in an incubator or subcutaneously in rats for 10, 20, and 30 days before immersion in methylene blue dye for 24 hours. Linear dye penetration was evaluated, and the results were statistically analyzed by means of analysis of variance. RESULTS: Leakage between sheep and human teeth was significantly different (P <.05). Chemfil had significantly less leakage than intermediate restorative material after storage in rat (P <.05) for up to 20 days, but not after 30 days. No differences were found between leakage of unfilled and filled human root canal teeth. CONCLUSIONS: The sheep incisor is a poor experimental model of the human tooth, and both aging procedures demonstrate extensive leakage of retrofilling materials after long-term storage.     

Psychological factors in people at ultra-high risk of psychosis: comparisons with non-patients and associations with symptoms

BACKGROUND: There have been recent advances in the ability to identify people at high risk of developing psychosis. This has led to interest in the possibility of preventing the development of psychosis and provides the opportunity to investigate psychological mechanisms that may confer vulnerability to psychosis. METHOD: Fifty-eight patients at ultra-high risk of developing a first episode of psychosis were compared with 56 non-patients matched for age and occupational status on measures of meta-cognition, schizotypal traits, dysfunctional attitudes and distress. RESULTS: Analyses of covariance revealed that people at high risk of developing psychosis scored higher on measures of cognitive vulnerability, including negative meta-cognitive beliefs, beliefs about rejection and criticism from others, and discrepancies in self-perception, schizotypal traits and general mental distress. Correlational analyses revealed that negative meta-cognitive beliefs, dysfunctional attitudes and beliefs about rejection and criticism from others were positively associated with several dimensions of symptomatology in at-risk mental states (ARMS) patients. CONCLUSIONS: Cognitive and personality factors appear to characterize people at high-risk of developing psychosis and are associated with their distressing experiences. The clinical implications of these findings are discussed.     

Hypertension and dementia

Hypertension is one of the principal risk factors for cerebrovascular diseases. Several epidemiologic studies have also indicated a positive correlation between cognitive decline or dementia and blood pressure level. Indeed, the results of most longitudinal studies show that cognitive functioning is often inversely proportional to blood pressure values measured 15 or 20 years previously. Cerebral infarcts, lacunae, and white matter changes are implicated in the pathogenesis of vascular dementia, but may also favor the development of Alzheimer’s disease. Microcirculation disorders and endothelial dysfunctions are also advanced to explain the deterioration in cognitive functions in hypertensive subjects. Data from recent therapeutic trials open the way to the prevention of dementia (vascular or Alzheimer’s type) by antihypertensive treatments and must be confirmed by other studies.     

Primary prophylaxis with granulocyte colony-stimulating factor (GCSF) reduces the incidence of febrile neutropenia in patients with non-Hodgkin lymphoma (NHL) receiving CHOP chemotherapy treatment without adversely affecting their quality of life: cost–benefit and quality of life analysis

Purpose

Treatment of non-Hodgkin lymphoma (NHL) with cyclophosphamide, vincristine, doxorubicin and prednisone (CHOP) is known to be associated with a significant risk of febrile neutropenia (FN) of up to 50 % [Osby et al. 2003 Blood 101(10): 3840–3848; Lyman and Delgado 2003 Cancer 98(11): 2402–2409]. This study sought to examine the impact of primary granulocyte colony-stimulating factor (GCSF) prophylaxis on the incidence of FN, quality of life and overall cost.

Methods

In this retrospective cohort study, a group of 65 consecutive patients who received CHOP chemotherapy for NHL between December 2006 and October 2009 was studied. Patients either received filgrastim (300 mcg, average of seven doses), pegylated filgrastim (6 mg, single dose), or no GCSF prophylaxis. In addition, 19 patients were asked to complete Functional Assessment of Cancer Therapy: General quality-of-life questionnaires.

Results

Overall, patients who received primary GCSF prophylaxis had significantly fewer FN compared to those who did not (5 vs. 60 %, p?<?0.0001; numbers needed to treat of 1.8; 95 % confidence interval, 1.6–2.9). Cost–benefit analysis showed that the GCSF prophylaxis was associated with only a small increase in direct financial cost ($238 NZD [US$189] more to give primary GCSF prophylaxis per patient vs. no prophylaxis). The quality of life assessment showed that the patients’ quality of life scores were similar to the published data from the validation study population (466 patients with mixed cancers) for Functional Assessment of Cancer Therapy.

Conclusions

Our study shows that primary GCSF prophylaxis is effective in preventing FN in patients receiving CHOP chemotherapy for NHL without adversely affecting their quality of life, and is cost effective.     

Infective Endocarditis and Septic Embolization with Ochrobactrum anthropi: Case Report and Review of Literature

Ochrobactrum anthropi, previously known as CDC group Vd, is an aerobic, Gram-negative bacillus of low virulence that occasionally causes human infection. We describe a case of infective endocarditis with O. anthropi complicated by septic embolization. A review of all the literature reported cases of O. anthropi infection is presented and categorized into ‘Central line related’, ‘Transplant related’ and ’Other pyogenic infections’. Mortality appears to be related to the underlying disease state, rather than the organism.