Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding

ATP-sensitive potassium (K(ATP)) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K(+) channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved "signature sequence" (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 microM) to displace 8-azido-[(32)P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced K(ATP) current activation by 100 microM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-R50G co-expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.     

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.     

New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel mutation underlies both syndromes

Mutations of CIAS1 have recently been shown to underlie familial cold urticaria (FCU) and Muckle-Wells syndrome (MWS), in three families and one family, respectively. These rare autosomal dominant diseases are both characterized by recurrent inflammatory crises that start in childhood and that are generally associated with fever, arthralgia, and urticaria. The presence of sensorineural deafness that occurs later in life is characteristic of MWS. Amyloidosis of the amyloidosis-associated type is the main complication of MWS and is sometimes associated with FCU. In FCU, cold exposure is the triggering factor of the inflammatory crisis. We identified CIAS1 mutations, all located in exon 3, in nine unrelated families with MWS and in three unrelated families with FCU, originating from France, England, and Algeria. Five mutations--namely, R260W, D303N, T348M, A439T, and G569R--were novel. The R260W mutation was identified in two families with MWS and in two families with FCU, of different ethnic origins, thereby demonstrating that a single CIAS1 mutation may cause both syndromes. This result indicates that modifier genes are involved in determining either a MWS or a FCU phenotype. The finding of the G569R mutation in an asymptomatic individual further emphasizes the importance of such modifier a gene (or genes) in determining the disease phenotype. Identification of this gene (or these genes) is likely to have significant therapeutic implications for these severe diseases.     

The effect of low-molecular-weight heparin in the survival of a rabbit congested skin flap

Swelling and congestion of flaps are frequently seen postoperatively and can cause unexpected necrosis. According to previous reports, venous thrombosis seems to be a more frequent problem than arterial occlusion in both experimental and clinical surgery. Few satisfactory venous trauma models exist, and reports on experimental venous thrombosis are rare. The object of this study was to create a rabbit venous occlusion flap model and to evaluate the effect of low-molecular-weight heparin on this flap. Eight New Zealand rabbits were used in the pilot study, in which the ideal congested flap was investigated using a flap pedicle based on the central auricular artery with a skin pedicle 0, 1, 2, or 3 cm wide. The flap (3 x 6 cm) was designed on the central part of the left ear, and the central auricular vein and nerve, the former for venous return, were cut out at the base of the flap. The flaps with skin pedicles 0, 1, 2, or 3 cm wide showed mean necrosis length of 60.0, 9.3, 4.2, and 0.0 mm, respectively. The flaps with skin pedicles 0, 1, 2, or 3 cm wide showed mean necrosis of 100, 15.5, 7, and 0 percent, respectively. Therefore, the flap, based on a 1-cm-wide skin pedicle and the central auricular artery, was selected as an optimal congested flap model showing 15.5 percent necrosis. The congested flap was then elevated on the left ear of another 10 rabbits. Subcutaneous low-molecular-weight heparin (320 IU/kg) was administered immediately after surgery to five of the rabbits (the low-molecular-weight heparin group), and the remaining five were used as a control group. Fluorescein was injected 15 minutes after surgery to evaluate the circulatory territory of the flap, and the circulatory territory was measured 5 minutes after injection. The flaps were assessed 7 days after surgery by angiography, histology, and clinical findings. The circulatory territory was significantly greater in the low-molecular-weight heparin group (mean +/- SD, 39.2 +/- 3.0 mm) than the control group (mean +/- SD, 48.0 +/- 1.0 mm) (p < 0.001) assessed 7 days after surgery. The longest flap survival length in group A and group B ranged from 40 to 55 mm (mean +/- SD. 49.4 +/- 5.6 mm) and complete survival (mean +/- SD, 60.0 +/- 0.0 mm). The improvement in survival was statistically significant for group B compared with group A (p < 0.015). Histologic evaluation revealed moderate to severe venous congestion and inflammation in the control group, whereas there were minimal changes in the low-molecular-weight heparin group. Angiography of the flap revealed obvious venous occlusion in the periphery in the control group compared with the low-molecular-weight heparin group. The authors conclude that subcutaneous administration of low-molecular-weight heparin has a great potential to improve the survival length of a congested flap without major complications.     

Gain modulation from background synaptic input

Gain modulation is a prominent feature of neuronal activity recorded in behaving animals, but the mechanism by which it occurs is unknown. By introducing a barrage of excitatory and inhibitory synaptic conductances that mimics conditions encountered in vivo into pyramidal neurons in slices of rat somatosensory cortex, we show that the gain of a neuronal response to excitatory drive can be modulated by varying the level of "background" synaptic input. Simultaneously increasing both excitatory and inhibitory background firing rates in a balanced manner results in a divisive gain modulation of the neuronal response without appreciable signal-independent increases in firing rate or spike-train variability. These results suggest that, within active cortical circuits, the overall level of synaptic input to a neuron acts as a gain control signal that modulates responsiveness to excitatory drive.     

Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides

Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.     

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction.     

Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E

A selective disruption of the mouse CENP-E gene was generated to test how this kinetochore-associated, kinesin-like protein contributes to chromosome segregation. The removal of CENP-E in primary cells produced spindles in which some metaphase chromosomes lay juxtaposed to a spindle pole, despite the absence of microtubules stably bound to their kinetochores. Most CENP-E-free chromosomes moved to the spindle equator, but their kinetochores bound only half the normal number of microtubules. Deletion of CENP-E in embryos led to early developmental arrest. Selective deletion of CENP-E in liver revealed that tissue regeneration after chemical damage was accompanied by aberrant mitoses marked by chromosome missegregation. CENP-E is thus essential for the maintenance of chromosomal stability through efficient stabilization of microtubule capture at kinetochores.     

Evidence for cAMP-dependent protein kinase in the dinoflagellate,Amphidinium operculatum

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.