Functional coupling between the Kv1.1 channel and aldoketoreductase Kvbeta1

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.     

Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that "activation" of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine omega-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS(-/-) mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.     

Temporal requirement of the alternative-splicing factor Sfrs1 for the survival of retinal neurons

Alternative splicing is the primary mechanism by which a limited number of protein-coding genes can generate proteome diversity. We have investigated the role of the alternative-splicing factor Sfrs1, an arginine/serine-rich (SR) protein family member, during mouse retinal development. Loss of Sfrs1 function during embryonic retinal development had a profound effect, leading to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early to mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. By contrast, Sfrs1 was not required for the survival of the neurons generated later, including later-born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated alternative splicing for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated.     

Retinoic acid-induced alveolar cellular growth does not improve function after right pneumonectomy.

To determine whether all-trans retinoic acid (RA) treatment enhances lung function during compensatory lung growth in fully mature animals, adult male dogs (n = 4) received 2 mg x kg(-1) x day(-1) po RA 4 days/wk beginning the day after right pneumonectomy (R-PNX, 55-58% resection). Litter-matched male R-PNX controls (n = 4) received placebo. After 3 mo, transpulmonary pressure (TPP)-lung volume relationship, diffusing capacities for carbon monoxide and nitric oxide, cardiac output, and septal volume (V(tiss-RB)) were measured under anesthesia by a rebreathing technique at two lung volumes. Lung air and tissue volumes (V(air-CT) and V(tiss-CT)) were also measured from high-resolution computerized tomographic (CT) scans at a constant TPP. In RA-treated dogs compared with controls, TPP-lung volume relationships were similar. Diffusing capacities for carbon monoxide and nitric oxide were significantly impaired at a lower lung volume but similar at a high lung volume. Whereas V(tiss-RB) was significantly lower at both lung volumes in RA-treated animals, V(air-CT) and V(tiss-CT) were not different between groups; results suggest uneven distribution of ventilation consistent with distortion of alveolar geometry and/or altered small airway function induced by RA. We conclude that RA does not improve resting pulmonary function during the early months after R-PNX despite histological evidence of its action in enhancing alveolar cellular growth in the remaining lung.     

Comparative study of catalase-peroxidases (KatGs)

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus,Bacillus stearothermophilus, Burkholderia pseudomallei, Escherichia coli, Mycobacterium tuberculosis, Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s⁻¹ and 8-25 s⁻¹, respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25-5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC₅₀ values of 0.2-20 μM and 50-170 μM, respectively. The K_Ms of the enzymes for H₂O₂ in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H₂O₂ binding to Cpd I. The catalatic k_cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s⁻¹, respectively. Only the NADH oxidase reaction varied more widely between 10⁻⁴ and 10⁻² s⁻¹ with the fastest rate being exhibited by the enzyme from B. pseudomallei.     

Identification of candidate regulators of multipotency in human skeletal progenitor cells

Stem cell differentiation is controlled intrinsically by dynamic networks of interacting lineage-specifying and multipotency genes. However, the relationship between internal genetic dynamics and extrinsic regulation of internal dynamics is complex and, in the case of skeletal progenitor cell differentiation, incompletely understood. In this study we elucidate a set of candidate markers of multipotency in human skeletal progenitor cells by systematic study of the relationships between gene expression and environmental stimulus. We used full genome cDNA microarrays to explore gene expression profiles in skeletal progenitor enriched populations derived from adult human bone marrow, minimally cultured in basal, osteogenic, chondrogenic, and adipogenic lineage-specifying culture conditions. We then used a variety of statistical clustering procedures to identify a small subset of genes which are related to these stromal lineages but are specific to none. For a selection of 11 key genes, conclusions of the microarray study were confirmed using quantitative real-time PCR.     

Control of Neuronal Network in Caenorhabditis elegans

Caenorhabditis elegans, a soil dwelling nematode, is evolutionarily rudimentary and contains only ∼ 300 neurons which are connected to each other via chemical synapses and gap junctions. This structural connectivity can be perceived as nodes and edges of a graph. Controlling complex networked systems (such as nervous system) has been an area of excitement for mankind. Various methods have been developed to identify specific brain regions, which when controlled by external input can lead to achievement of control over the state of the system. But in case of neuronal connectivity network the properties of neurons identified as driver nodes is of much importance because nervous system can produce a variety of states (behaviour of the animal). Hence to gain insight on the type of control achieved in nervous system we implemented the notion of structural control from graph theory to C. elegans neuronal network. We identified ‘driver neurons’ which can provide full control over the network. We studied phenotypic properties of these neurons which are referred to as ‘phenoframe’ as well as the ‘genoframe’ which represents their genetic correlates. We find that the driver neurons are primarily motor neurons located in the ventral nerve cord and contribute to biological reproduction of the animal. Identification of driver neurons and its characterization adds a new dimension in controllability of C. elegans neuronal network. This study suggests the importance of driver neurons and their utility to control the behaviour of the organism.