Enhanced β(2)-adrenergic receptor (β(2)AR) signaling by adeno-associated viral (AAV)-mediated gene transfer

BACKGROUND: β(2)-Adrenergic receptors (β(2)AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of β-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the β(2)AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-β(2)AR/EGFP). RESULTS: By epifluorescence microscopy, ~40% of infected HEK 293 cells demonstrated EGFP expression. β(2)AR density measured with [(3)H]dihydroalprenolol ([(3)H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [(3)H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type β(2)AR. Agonist competition assays with [(3)H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with β(2)AR interaction. Isoproterenol-stimulated cyclic AMP levels were 5-fold higher in infected cells compared to controls (314 ± 43 vs. 63.4 ± 9.6 nmol/dish; n = 3). Receptor trafficking demonstrated surface expression of β(2)AR with vehicle treatment and internalization following isoproterenol treatment. CONCLUSIONS: We conclude that HEK 293 cells infected with AAV-β(2)AR/EGFP effectively express β(2)AR and that increased expression of these receptors results in enhanced β(2)AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems

XMM-Newton observations of the supernova remnant IC443: I. soft X-ray emission from shocked interstellar medium

The shocked interstellar medium around IC443 produces strong X-ray emission in the soft energy band (E<1.5 keV). We present an analysis of such emission as observed with the EPIC MOS cameras on board the XMM-Newotn observatory, with the purpose to find clear signatures of the interactions with the interstellar medium (ISM) in the X-ray band, which may complement results obtained in other wavelenghts. We found that the giant molecular cloud mapped in CO emission is located in the foreground and gives an evident signature in the absorption of X-rays. This cloud may have a torus shape and the part of torus interacting with the IC443 shock gives rise to 2MASS-K emission in the southeast. The measured density of emitting X-ray shocked plasma increases toward the northeastern limb, where the remnant is interacting with an atomic cloud. We found an excellent correlation between emission in the 0.3-0.5 keV band and bright optical/radio filament on large spatial scales. The partial shell structure seen in this band therefore traces the encounter with the atomic cloud.Comment: 10 pages, 10 figures, accepted for publication in ApJ (20 September 2006, v649). For hi-res figures, see http://www.astropa.unipa.it/Library/OAPA_preprints/ic443ele1.ps.g

Preliminary analysis of an extensive one year survey of trace elements and compounds in the suspended particulate matter in Cleveland, Ohio

Beginning in 1971 a cooperative program has been carried on by the City of Cleveland Division of Air Pollution Control and NASA Lewis Research Center to study the trace element and compound concentrations in the ambient suspended particulate matter in Cleveland Ohio as a function of source, monitoring location and meteorological conditions. The major objectives were to determine the ambient concentration levels at representative urban sites and to develop a technique using trace element and compound data in conjunction with meteorological conditions to identify specific pollution sources which could be developed into a practical system that could be readily utilized by an enforcement agency

Phencyclidine Continuous Dosing Produces a Treatment Time- Dependent Regulation of Rat CYP2C11 Function, Protein Expression and mRNA Levels 1

ABSTRACT These studies determined the effects of continuous phencyclidine (PCP) administration on cytochrome P 450 2C11 (CYP2C11) function, protein expression and mRNA levels. Male Sprague-Dawley rats received s.c. PCP infusions (18 mg/kg/ day) for 1, 3, 10 or 20 days (n ϭ 4 per group). Control animals received saline infusions for 3 or 20 days. Livers were collected 24 hr postinfusion, a time when PCP was completely cleared from the animals. In microsomes from the 1-and 3-day PCP infusions, there was a significant decrease (P Ͻ .05) in CYP2C11 protein expression (61 and 46% of control values, respectively) and in CYP2C11-mediated metabolism of PCP to a reactive metabolite (36 and 41% of control values). Both protein expression and PCP metabolite formation had returned to normal by 10 days of continuous PCP infusion. CYP2C11 function (as measured by 2␣-OH testosterone formation) was decreased in the 1-, 3-and 10-day infused rats to 46, 28 and 45% of control values (P Ͻ .05). CYP2C11 function, expression and reactive PCP metabolite formation returned to normal after 20 days of PCP infusion. In contrast, CYP2C11 mRNA levels were decreased (P Ͻ .05) in liver tissue in PCP-treated rats from 1 to 20 days (43, 31, 37 and 47%, respectively). These data suggest that continuous PCP infusions initially decrease CYP2C11 function and protein expression by a pretranslational mechanism, but continued exposure to PCP leads to metabolic adaptation without the recovery of mRNA levels. Thus, chronic exposure to PCP can produce time-dependent regulation of CYP2C11-mediated metabolism of endogenous and exogenous compounds. PCP is metabolized by CYP enzymes to at least six major metabolites in mammals Chronic s.c. infusions of PCP for up to 20 days in normal male Sprague-Dawley rats produce complex time-and dosedependent changes in the in vitro metabolism of PC

Isolated X-ray -- infrared sources in the region of interaction of the supernova remnant IC 443 with a molecular cloud

The nature of the extended hard X-ray source XMMU J061804.3+222732 and its surroundings is investigated using XMM-Newton, Chandra, and Spitzer observations. This source is located in an interaction region of the IC 443 supernova remnant with a neighboring molecular cloud. The X-ray emission consists of a number of bright clumps embedded in an extended structured non-thermal X-ray nebula larger than 30" in size. Some clumps show evidence for line emission at ~1.9 keV and ~3.7 keV at the 99% confidence level. Large-scale diffuse radio emission of IC 443 passes over the source region, with an enhancement near the source. An IR source of about 14" x 7" size is prominent in the 24 um, 70 um, and 2.2 um bands, adjacent to a putative Si K-shell X-ray line emission region. The observed IR/X-ray morphology and spectra are consistent with those expected for J/C-type shocks of different velocities driven by fragmented supernova ejecta colliding with the dense medium of a molecular cloud. The IR emission of the source detected by Spitzer can be attributed to both continuum emission from an HII region created by the ejecta fragment and line emission excited by shocks. This source region in IC 443 may be an example of a rather numerous population of hard X-ray/IR sources created by supernova explosions in the dense environment of star-forming regions. Alternative Galactic and extragalactic interpretations of the observed source are also discussed.Comment: The Astrophysical Journal, v. 677 (April 2008), in pres

Characterization of a panel of six β2-adrenergic receptor antibodies by indirect immunofluorescence microscopy

<p>Abstract</p> <p>Background</p> <p>The β₂-adrenergic receptor (β₂AR) is a primary target for medications used to treat asthma. Due to the low abundance of β₂AR, very few studies have reported its localization in tissues. However, the intracellular location of β₂AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β₂AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β₂AR in primary cell cultures.</p> <p>Methods</p> <p>A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β₂AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β₂AR were identified and used to localize native β₂AR in primary cultures of rat airway smooth muscle and epithelial cells. β₂AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ^([3H]DHA).</p> <p>Results</p> <p>Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β₂AR (Ab-Bethyl) specifically recognized human but not rat β₂AR. An antibody developed against the C-terminal domain of the mouse β₂AR (Ab-sc570) specifically recognized rat but not human β₂AR. An antibody developed against 78 amino acids of the C-terminus of the human β₂AR (Ab-13989) was capable of recognizing both rat and human β₂ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β₂AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.</p> <p>Conclusion</p> <p>Antibodies have been identified that recognize human β₂AR, rat β₂AR or both rat and human β₂AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β₂AR in tissues.</p

Methamphetamine Use: A Narrative Review of Adverse Effects and Related Toxicities.

Methamphetamine has been labeled America\u27s most dangerous drug and has received significant public health attention. Stimulant addiction and tolerance are heavily documented in the literature; increasingly larger doses maintain euphoria in short time periods to withstand stimulant tolerance. Stimulant deaths are high in the United States and abroad. Between 2013 and 2019, deaths related to methamphetamine use quadrupled from 3,616 to 16,127. Methamphetamine use increased four-fold from 2015 to 2016. Due to this increase in methamphetamine use and its associated medical complications, the mortality rate associated with methamphetamine use has doubled over the past ten years. Cardiopulmonary symptoms include chest pain, palpitations, and shortness of breath. Methamphetamine-related myocardial infarction can also occur. Central nervous system symptoms include agitation, anxiety, delusions, hallucinations, and seizures. Methamphetamine-induced psychosis may unmask underlying psychiatric disorders. It can also cause cerebral vasculitis, which elicits cortical blindness and ischemic strokes. Methamphetamine-induced neurotoxicity in serotonergic systems is more diffuse, involving the striatum, hippocampus, septum, amygdala, and hypothalamus leading to mood changes, psychosis, and memory impairment. This narrative review will aim to highlight the adverse effects as well as the toxicity that can occur with methamphetamine use

Comparing Galaxy Morphology at Ultraviolet and Optical Wavelengths

We have undertaken an imaging survey of 34 nearby galaxies in far-ultraviolet (FUV, ~1500A) and optical (UBVRI) passbands to characterize galaxy morphology as a function of wavelength. This sample, which includes a range of classical Hubble types from elliptical to irregular with emphasis on spirals at low inclination angle, provides a valuable database for comparison with images of high-z galaxies whose FUV light is redshifted into the optical and near- infrared bands. Ultraviolet data are from the UIT Astro-2 mission. We present images and surface brightness profiles for each galaxy, and we discuss the wavelength-dependence of morphology for different Hubble types in the context of understanding high-z objects. In general, the dominance of young stars in the FUV produces the patchy appearance of a morphological type later than that inferred from optical images. Prominent rings and circumnuclear star formation regions are clearly evident in FUV images of spirals, while bulges, bars, and old, red stellar disks are faint to invisible at these short wavelengths. However, the magnitude of the change in apparent morphology ranges from dramatic in early--type spirals with prominent optical bulges to slight in late-type spirals and irregulars, in which young stars dominate both the UV and optical emission. Starburst galaxies with centrally concentrated, symmetric bursts display an apparent ``E/S0'' structure in the FUV, while starbursts associated with rings or mergers produce a peculiar morphology. We briefly discuss the inadequacy of the optically-defined Hubble sequence to describe FUV galaxy images and estimate morphological k-corrections, and we suggest some directions for future research with this dataset.Comment: Accepted for publication in the ApJS. 15 pages, 17 JPEG figures, 10 GIF figures. Paper and full resolution figures available at http://nedwww.ipac.caltech.edu/level5/Kuchinski/frames.htm

Antipsychotic Use in Pregnancy: Patient Mental Health Challenges, Teratogenicity, Pregnancy Complications, and Postnatal Risks

Pregnant women constitute a vulnerable population, with 25.3% of pregnant women classified as suffering from a psychiatric disorder. Since childbearing age typically aligns with the onset of mental health disorders, it is of utmost importance to consider the effects that antipsychotic drugs have on pregnant women and their developing fetus. However, the induction of pharmacological treatment during pregnancy may pose significant risks to the developing fetus. Antipsychotics are typically introduced when the nonpharmacologic approaches fail to produce desired effects or when the risks outweigh the benefits from continuing without treatment or the risks from exposing the fetus to medication. Early studies of pregnant women with schizophrenia showed an increase in perinatal malformations and deaths among their newborns. Similar to schizophrenia, women with bipolar disorder have an increased risk of relapse in antepartum and postpartum periods. It is known that antipsychotic medications can readily cross the placenta, and exposure to antipsychotic medication during pregnancy is associated with potential teratogenicity. Potential risks associated with antipsychotic use in pregnant women include congenital abnormalities, preterm birth, and metabolic disturbance, which could potentially lead to abnormal fetal growth. The complex decision-making process for treating psychosis in pregnant women must evaluate the risks and benefits of antipsychotic drugs

Benzodiazepines: Uses, dangers, and clinical considerations

Benzodiazepines (BZDs) are among one of the most widely prescribed drug classes in the United States. BZDs are a class of psychoactive drugs known for their depressant effect on the central nervous system (CNS). They quickly diffuse through the blood–brain barrier to affect the inhibitory neurotransmitter GABA and exert sedative effects. Related to their rapid onset and immediate symptom relief, BZDs are used for those struggling with sleep, anxiety, spasticity due to CNS pathology, muscle relaxation, and epilepsy. One of the debilitating side effects of BZDs is their addictive potential. The dependence on BZDs generally leads to withdrawal symptoms, requiring careful tapering of the medication when prescribed. Regular use of BZDs has been shown to cause severe, harmful psychological and physical dependence, leading to withdrawal symptoms similar to that of alcohol withdrawal. Some of these withdrawal symptoms can be life threatening. The current treatment for withdrawal is through tapering with clonazepam. Many drugs have been tested as a treatment for withdrawal, with few proving efficacious in randomized control trials. Future research is warranted for further exploration into alternative methods of treating BZD withdrawal. This call to action proves especially relevant, as those seeking treatment for BZD dependence and withdrawal are on the rise in the United States