The relationship between apamin binding and channel block in KCa2 potassium channels.

Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are widely distributed throughout the body and are involved in diverse physiological processes including the regulation of neuronal firing and smooth muscle contraction. They are also potential targets in the treatment of cardiac arrhythmia. The KCa2.2 and 2.3 members of the family are blocked by the peptide toxin apamin at low concentrations, however, the mechanism of block by apamin is unclear. In electrophysiological experiments apamin has been reported to block Kca2.2 and 2.3 with IC50 ~100 pM and ~1nM respectively. In contrast, in ligand binding experiments using [125I]-mono-iodoapamin it has been found that apamin does not discriminate between Kca2.2 and 2.3 and that it binds with significantly higher affinity ( ~5pM). This discrepancy has led to the suggestion that, rather than acting as a classical pore blocker, apamin exerts its action by an allosteric mechanism. It is notable that the ligand binding studies reported so far have been conducted with isolated cell membranes in non-physiological solution with low ionic strength. We have investigated this discrepancy between results from ligand binding and electrophysiological studies by comparing binding of [125I]-mono-iodoapamin and inhibition of KCa2 current in intact HEK 293 cells using identical physiological solutions. In these conditions we found that apamin bound to KCa2.1 and KCa 2.3 with KL 60 and 606 pM, close to values of IC50 from electrophysiological experiments. We also compared the ability of some known SK channel blockers, UCL 1848, UCL 1684, gallamine and dequalinium, to displace labelled apamin and inhibit KCa2 current. With these compounds we found a good correlation between K¬i and IC50. These findings suggest that the discrepancy between binding and block might arise from differences in the experimental protocols used. To examine this we examined apamin block of KCa2 current in low ionic strength solutions in which NaCl was iso-osmotically replaced by sucrose. In these conditions 100 pM apamin caused 92 ± 0.1 % block as against 51 ± 5 % block in physiological ionic strength. We conclude that binding data obtained from membrane preparations must be interpreted with care when making comparisons with data from functional experiments and that this has implications for current views on the mechanism of action of apamin as an SK channel blocke

Selective inhibition of T suppressor-cell function by a monosaccharide

Interactions between regulatory T lymphocytes and other cells are assumed to occur at the level of the cell surface. T cells which suppress the generation of specifically effector cells have been described as having antigenic, idiotypic, allotypic and I-region specificity1−4. Other T suppressor cells generated by in vitro cultivation with or without mitogenic stimulation5,6 have suppressive activity for T and B cells but no specificity can be assigned to them. These T suppressor cells (Ts) inhibit various lymphoid functions—this either reflects their polyclonal origin or indicates that the structures recognized by the Ts receptors must be common for many cell types. Carbohydrates on cell membrane-inserted glycoproteins or glycolipids might function as specific ligands for recognition by cellular receptors or soluble factors. Almost all cell-surface proteins of mammalian cells are glycosylated. There is evidence for lectin-like carbohydrate binding proteins not only in plants7 but also in toxins8, viruses9, prokaryotic cells10 and even mammalian cells, including T cells11. A functional role for these lectin-like proteins has been described for slime moulds and suggested for the selective association of embryonic cells12,13. We report here that addition of a monosaccharide can counteract the effect of T suppressor cells during the generation of alloreactive cytotoxic T cells (CTLs) in vitro

FAN1 modifies Huntington's disease progression by stabilising the expanded HTT CAG repeat

Huntington's disease (HD) is an inherited neurodegenerative disease caused by an expanded CAG repeat in the HTT gene. CAG repeat length explains around half of the variation in age-at-onset, but genetic variation elsewhere in the genome accounts for a significant proportion of the remainder. Genome-wide association studies have identified a bidirectional signal on chromosome 15, likely underlain by FAN1 (FANCD2 and FANCI Associated Nuclease 1), a nuclease involved in DNA interstrand cross link repair. Here we show that increased FAN1 expression is significantly associated with delayed age-at-onset and slower progression of HD suggesting FAN1 is protective in the context of an expanded HTT CAG repeat. FAN1 overexpression in human cells reduces CAG repeat expansion in exogenously expressed mutant HTT exon 1, and in patient-derived stem cells and differentiated medium spiny neurons, FAN1 knockdown increases CAG repeat expansion. The stabilising effect is FAN1 concentration and CAG repeat length dependent. We show that FAN1 binds to the expanded HTT CAG repeat DNA and its nuclease activity is not required for protection against CAG repeat expansion. These data shed new mechanistic insights into how the genetic modifiers of HD act to alter disease progression, and show that FAN1 affects somatic expansion of the CAG repeat through a nuclease-independent mechanism. This provides new avenues for therapeutic interventions in HD and potentially other triplet repeat disorders

Influenza A Virus Coding Regions Exhibit Host-Specific Global Ordered RNA Structure

Influenza A is a significant public health threat, partially because of its capacity to readily exchange gene segments between different host species to form novel pandemic strains. An understanding of the fundamental factors providing species barriers between different influenza hosts would facilitate identification of strains capable of leading to pandemic outbreaks and could also inform vaccine development. Here, we describe the difference in predicted RNA secondary structure stability that exists between avian, swine and human coding regions. The results predict that global ordered RNA structure exists in influenza A segments 1, 5, 7 and 8, and that ranges of free energies for secondary structure formation differ between host strains. The predicted free energy distributions for strains from avian, swine, and human species suggest criteria for segment reassortment and strains that might be ideal candidates for viral attenuation and vaccine development

A blind accuracy assessment of computer-modeled forensic facial reconstruction using computed tomography data from live subjects.

A computer modeling system for facial reconstruction has been developed that employs a touch-based application to create anatomically accurate facial models focusing on skeletal detail. This article discusses the advantages and disadvantages of the system and illustrates its accuracy and reliability with a blind study using computed tomography (CT) data of living individuals. Three-dimensional models of the skulls of two white North American adults (one male, one female) were imported into the computer system. Facial reconstructions were produced by two practitioners following the Manchester method. Two posters were produced, each including a face pool of five surface model images and the facial reconstruction. The face pool related to the sex, age, and ethnic group of the target individual and included the surface model image of the target individual. Fifty-two volunteers were asked to choose the face from the face pool that most resembled each reconstruction. Both reconstructions received majority percentage hit rates that were at least 50% greater than any other face in the pool. The combined percentage hit rate was 50% above chance (70%). A quantitative comparison of the facial morphology between the facial reconstructions and the CT scan models of the subjects was carried out using Rapidform(™) 2004 PP2-RF4. The majority of the surfaces of the facial reconstructions showed less than 2.5 mm error and 90% of the male face and 75% of the female face showed less than 5 mm error. Many of the differences between the facial reconstructions and the facial scans were probably the result of positional effects caused during the CT scanning procedure, especially on the female subject who had a fatter face than the male subject. The areas of most facial reconstruction error were at the ears and nasal tip

Intravitreal vs. subtenon triamcinolone acetonide for the treatment of diabetic cystoid macular edema

<p>Abstract</p> <p>Background</p> <p>To assess the efficacy of the intravitreal (IVT) injection of Triamcinolone Acetonide (TA) as compared to posterior subtenon (SBT) capsule injection for the treatment of cystoid diabetic macular edema.</p> <p>Methods</p> <p>Fourteen patients with type II diabetes mellitus and on insulin treatment, presenting diffuse cystoid macular edema were recruited. Before TA injection all focal lakes were treated by laser photocoagulation. In the same patients one eye was assigned to 4 mg IVT injection of TA and the fellow eye was then treated with 40 mg SBT injection of TA. Before and one, three and six months after treatment we measured visual acuity with ETDRS chart as well as thickness of the macula with optical coherence tomography (OCT) and intraocular pressure (IOP).</p> <p>Results</p> <p>The eyes treated with an IVT injection displayed significant improvement in visual acuity, both after one (0.491 ± 0.070; p < 0.001) and three months (0.500 ± 0.089; p < 0.001) of treatment. Significant improvement was displayed also in eyes treated with an SBT injection, again after one (0.455 ± 0.069; p < 0.001) and three months (0.427 ± 0.065; p < 0.001). The difference between an IVT injection (0.809 ± 0.083) and SBT injection (0.460 ± 0.072) becomes significant six months after the treatment (p < 0.001).</p> <p>Macular thickness of the eyes treated with IVT injection was significantly reduced both after one (222.7 ± 13.4 μm; p < 0.001) and after three months (228.1 ± 10.6 μm; p < 0.001) of treatment. The eyes treated with SBT injection displayed significant improvement after one (220.1 ± 15.1 μm; p < 0.001) and after three months (231.3 ± 10.9 μm; p < 0.001). The difference between the eyes treated with IVT injection (385.2 ± 11.3 μm) and those treated with SBT injection (235.4 ± 8.7 μm) becomes significant six months after the treatment (p < 0.001).</p> <p>Intraocular pressure of the eyes treated with IVT injection significantly increased after one month (17.7 ± 1.1 mm/Hg; p < 0.020), three (18.2 ± 1.2 mm/Hg; p < 0.003) and six month (18.1 ± 1.3 mm/Hg; p < 0.007) when compared to baseline value (16.1 ± 1.402 mm/Hg). In the SBT injection eyes we didn't display a significant increase of intraocular pressure after one (16.4 ± 1.2 mm/Hg; p < 0.450), three (16.3 ± 1.1 mm/Hg; p < 0.630) and six months (16.2 ± 1.1 mm/Hg; p < 0.720) when compared to baseline value (16.2 ± 1.3 mm/Hg).</p> <p>Conclusion</p> <p>The parabulbar subtenon approach can be considered a valid alternative to the intravitreal injection.</p> <p>Trial registration</p> <p>Current Controlled Trials <b>ISRCTN67086909</b></p

Genome Sequence of Erythromelalgia-Related Poxvirus Identifies it as an Ectromelia Virus Strain

Erythromelagia is a condition characterized by attacks of burning pain and inflammation in the extremeties. An epidemic form of this syndrome occurs in secondary students in rural China and a virus referred to as erythromelalgia-associated poxvirus (ERPV) was reported to have been recovered from throat swabs in 1987. Studies performed at the time suggested that ERPV belongs to the orthopoxvirus genus and has similarities with ectromelia virus, the causative agent of mousepox. We have determined the complete genome sequence of ERPV and demonstrated that it has 99.8% identity to the Naval strain of ectromelia virus and a slighly lower identity to the Moscow strain. Small DNA deletions in the Naval genome that are absent from ERPV may suggest that the sequenced strain of Naval was not the immediate progenitor of ERPV

lin-28 Controls the Succession of Cell Fate Choices via Two Distinct Activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7–independent positive regulation of hbl-1 through its 3′UTR to control L2 stage-specific cell fates; and second, a let-7–dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession

Providing alcohol-related screening and brief interventions to adolescents through health care systems: Obstacles and solutions

Duncan Clark and Howard Moss identify obstacles to alcohol-related screening and treatment for adolescents and propose policy solutions