Cardiac myofibroblasts differentiated in 3D culture exhibit distinct changes in collagen I production, processing, and matrix deposition

Myofibroblasts are a differentiated fibroblast cell type characterized by increased contractile capacity and elevated production of extracellular matrix (ECM) proteins. In the heart, myofibroblast expression is implicated in fibrosis associated with pressure-overload hypertrophy, among other pathologies. Although enhanced expression of ECM proteins by myofibroblasts is established, few studies have addressed the nature of the ECM deposited by myofibroblasts. To characterize ECM production and assembly by cardiac myofibroblasts, we developed a three-dimensional (3D) culture system using primary cardiac fibroblasts seeded into a nylon mesh that allows us to reversibly interconvert between myofibroblast and fibroblast phenotypes. We report that an increase in collagen I production by myofibroblasts was accompanied by a significant increase in collagen deposition into insoluble ECM. Furthermore, myofibroblasts exhibited increased levels of procollagen alpha1(I) with C-propeptide attached (and N-propeptide removed) relative to procollagen alpha1(I) compared with fibroblast cultures. An increase in production of the myofibroblast-associated splice variant of fibronectin (EDA-Fn) was seen in myofibroblast 3D cultures. Because the regulation of procollagen I processing is known to have profound effects on ECM assembly, differences in procollagen I secretion and maturation coupled with expression of EDA-Fn are shown to contribute to the production of a distinct ECM by the cardiac myofibroblast.     

Time-sharing in pigeons: Independent effects of gap duration, position and discriminability from the timed signal

Previous data suggest that in a peak-interval procedure with gaps, memory for the pre-gap interval varies with the discriminability of the gap from the to-be-timed signal. Here we extend this finding by manipulating the pre-gap and gap intervals as well as the visual contrast between the gap and the to-be-timed signal. The delay in response function after the gap was found to vary with the duration and position of the gap. However, for each gap duration and position, the delay in response increased with the gap-signal contrast: at 60% gap-signal contrast pigeons continued to accumulate time during the gap, at 80% gap-signal contrast pigeons stopped timing during the gap, and at 100% gap-signal contrast pigeons reset their timing after the gap. Data are accounted for by a time-sharing model assuming two concurrent processes during the gap--time accumulation and memory decay controlled by the salience of the gap--whose interplay results in a continuum of responses in the gap procedure.     

Enhancement of the recycling and activation of beta-adrenergic receptor by Rab4 GTPase in cardiac myocytes

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous beta-adrenergic receptor (beta-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. Beta1-AR, the predominant subtype of beta-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized beta-AR to the cell surface and enhanced beta-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of beta-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the beta-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous beta-AR and augmentation of Rab4-mediated traffic enhances beta-AR function in cardiac myocytes.     

The beta4-beta8 groove is an ATP-interactive site in the alpha crystallin core domain of the small heat shock protein, human alphaB crystallin

The site for ATP interactions in human alphaB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the alphaB crystallin sequence, (82)KHFSPEELKVKVLGD(96) as a Walker-B ATP-binding motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[gamma]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human alphaB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the beta4-beta8 groove as the ATP interactive site in alphaB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the beta8 strand ((131)LTITSSLS(138)). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the alphaB crystallin Walker-B motif photo-affinity-labeled with azido-ATP-EDANS confirming the beta4-beta8 groove as an ATP interactive site. The results presented here clearly establish the beta4-beta8 groove as the ATP interactive region in alphaB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.     

Sensitive Detection of Organophosphorus Pesticides Using a Needle Type Amperometric Acetylcholinesterase-based Bioelectrode. Thiocholine Electrochemistry and Immobilised Enzyme Inhibition

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl sat was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm ^?2 of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 μM for dipterex and 0.4 μM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.     

Δ9-THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

To characterize the long-term effects of adolescent marijuana abuse, we performed a proteomic analysis of cerebellar extracts from adult female rats with and without ovariectomy that were treated with Δ9-THC for 40 days during adolescence. Six proteins were found to significantly differ among the four treatment groups, with Δ9-THC and ovariectomy (OVX) decreasing the mitochondrial proteins, pyruvate carboxylase and NADH dehydrogenase, whereas the levels of putative cytosolic molecular chaperones NM23B, translationally controlled tumor protein, DJ-1 and activator of heat-shock 90kDa protein ATPase homolog 1 (AHA1) were increased. We further analyzed the effects of AHA1, a HSP90 co-chaperone, on CB1R and CB2R trafficking and signaling in transfected HEK293T and Neuro-2A cells. In HEK293T cells, AHA1 over-expression enhanced plasma membrane levels of CB1R and increased CB1R-mediated effects on cAMP levels and on MAPK phosphorylation. AHA1 over-expression also enhanced cell surface levels of endogenous CB1R and the effects of Δ9-THC on the cAMP levels in Neuro-2A cells. In contrast, over-expression of AHA1 did not affect the subcellular localization and signaling of CB2R. Our data indicate that chronic Δ9-THC administration in adolescence altered the endogenous levels of specialized proteins in the cerebellum, such as AHA1, and that this protein can change CB1R cell surface levels and signaling.     

Serodiagnosis of environmental mycobacterial infections

To demonstrate the usefulness of enzyme-linked immunosorbent assay for serodiagnosis of mycobacterioses due to environmental mycobacteria we utilized a panel of glycolipid antigens selective for Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium scrofulaceum and Mycobacterium gordonae. The levels of circulating antibodies were determined against the environmental mycobacteria, and Mycobacterium tuberculosis in human immunodeficiency virus-negative and -positive patient sera. The method used immunomagnetic separation of the antigens, with covalent immobilization of antibodies to superparamagnetic amine and carboxyl terminated particles in solutions of the specific antigens. Enzyme-linked immunosorbent assay was performed on 195 patient sera: 34 with infections due to environmental mycobacteria, 114 with tuberculosis, 47 with other respiratory diseases. There were 46 human immunodeficiency virus-1 infected individuals. Among the 34 infections due to environmental mycobacteria, 9 patients were singularly infected with an environmental mycobacterium, and 25 co-infected with both M. tuberculosis and an environmental mycobacterium. Sensitivity, specificity and false positivity ranges were determined for each of the volunteer groups: tuberculosis positive, human immunodeficiency virus negative; tuberculosis positive, human immunodeficiency virus positive; those with infections due to individual environmental mycobacteria (such as M. scrofulaceum and M. kansasii); and those with other respiratory diseases. We demonstrate that such multiple assays, can be useful for the early diagnosis of diverse environmental mycobacterial infections to allow the start of treatment earlier than henceforth.     

Neuroregeneration in neurodegenerative disorders

Background  

Neuroregeneration is a relatively recent concept that includes neurogenesis, neuroplasticity, and neurorestoration - implantation of viable cells as a therapeutical approach.     

Multiphoton fluorescence microscopy with GRIN objective aberration correction by low order adaptive optics

Graded Index (GRIN) rod microlenses are increasingly employed in the assembly of optical probes for microendoscopy applications. Confocal, two-photon and optical coherence tomography (OCT) based on GRIN optical probes permit in-vivo imaging with penetration depths into tissue up to the centimeter range. However, insertion of the probe can be complicated by the need of several alignment and focusing mechanisms along the optical path. Furthermore, resolution values are generally not limited by diffraction, but rather by optical aberrations within the endoscope probe and feeding optics. Here we describe a multiphoton confocal fluorescence imaging system equipped with a compact objective that incorporates a GRIN probe and requires no adjustment mechanisms. We minimized the effects of aberrations with optical compensation provided by a low-order electrostatic membrane mirror (EMM) inserted in the optical path of the confocal architecture, resulting in greatly enhanced image quality.