Detection of human and rodent 5-HT3B receptor subunits by anti-peptide polyclonal antibodies

Background: The 5-HT3 receptor is a member of a neurotransmitter-gated ion channel family which includes nicotinic acetylcholine, GABA(A), and glycine receptors. While antibodies specific for the 5-HT3A receptor subunit are plentiful, and have revealed a wealth of structural and functional information, few antisera exist for the detection of 5-HT3B receptor subunits. Here we describe the generation and characterisation of a rabbit polyclonal antiserum that specifically recognises 5HT(3B) receptor subunits|Results: Immunization of a rabbit with a 20-mer peptide, corresponding to the N-terminus of the human 5-HT3B receptor subunit, generated serum with polyclonal antibodies from which an IgG fraction was purified, yielding pAb77. The antibodies were shown to label 5-HT3B receptor subunits in transfected human embryonic kidney cells and rodent tissues using Western blots. Immunocytochemistry using pAb77 on these cells showed that 5-HT3B receptor subunits do not reach the plasma membrane in the absence of 5-HT3A receptor subunits. Immunohistochemical analysis of rat brain sections showed pAb77 immunoreactivity in distinct populations of cells in the hippocampus.|Conclusion: We have demonstrated that pAb77 antibodies specifically label native and recombinant 5-HT3B receptor subunits with high affinity and specificity. The antibody was shown to be useful for the determination of both receptor trafficking and also mapping 5-HT3B receptor subunit expression in the CNS

Estimating attraction basin sizes

The performance of local search algorithms is influenced by the properties that the neighborhood imposes on the search space. Among these properties, the number of local optima has been traditionally considered as a complexity measure of the instance, and different methods for its estimation have been developed. The accuracy of these estimators depends on properties such as the relative attraction basin sizes. As calculating the exact attraction basin sizes becomes unaffordable for moderate problem sizes, their estimations are required. The lack of techniques achieving this purpose encourages us to propose two methods that estimate the attraction basin size of a given local optimum. The first method takes uniformly at random solutions from the whole search space, while the second one takes into account the structure defined by the neighborhood. They are tested on different instances of problems in the permutation space, considering the swap and the adjacent swap neighborhoods

Loading rate and contraction duration effects on in vivo human Achilles tendon mechanical properties.

Tendons are viscoelastic, which implies loading rate dependency, but loading rates of contractions are often not controlled during assessment of human tendon mechanical properties in vivo. We investigated the effects of sustained submaximal isometric plantarflexion contractions, which potentially negate loading rate dependency, on the stiffness of the human Achilles tendon in vivo using dynamometry and ultrasonography. Maximum voluntary contractions (high loading rate), ramp maximum force contractions with 3 s loading (lower loading rate) and sustained contractions (held for 3 s) at 25%, 50% and 80% of maximal tendon force were conducted. No loading rate effect on stiffness (25-80% max. tendon force) was found. However, loading rate effects were seen up to 25% of maximum tendon force, which were reduced by the sustained method. Sustained plantarflexion contractions may negate loading rate effects on tendon mechanical properties and appear suitable for assessing human Achilles tendon stiffness in vivo

Does BCR/ABL1 positive Acute Myeloid Leukaemia Exist?

The BCR/ABL1 fusion gene, usually carried by the Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) or variants, is pathognomonic for chronic myeloid leukaemia (CML). It is also occasionally found in acute lymphoblastic leukaemia (ALL) mostly in adults and rarely in de novo acute myeloid leukaemia (AML). Array Comparative Genomic Hybridization (aCGH) was used to study six Ph(+)AML, three bi-lineage and four Ph(+)ALL searching for specific genomic profiles. Surprisingly, loss of the IKZF1 and/or CDKN2A genes, the hallmark of Ph(+)ALL, were recurrent findings in Ph(+)AML and accompanied cryptic deletions within the immunoglobulin and T cell receptor genes. The latter two losses have been shown to be part of 'hot spot' genome imbalances associated with BCR/ABL1 positive pre-B lymphoid phenotype in CML and Ph(+)ALL. We applied Significance Analysis of Microarrays (SAM) to data from the 'hot spot' regions to the Ph(+)AML and a further 40 BCR/ABL1(+) samples looking for differentiating features. After exclusion of the most dominant markers, SAM identified aberrations unique to de novo Ph(+)AML that involved relevant genes. While the biological and clinical significance of this specific genome signature remains to be uncovered, the unique loss within the immunoglobulin genes provides a simple test to enable the differentiation of clinically similar de novo Ph(+) AML and myeloid blast crisis of CML. © 2013 John Wiley & Sons Ltd and Crown

Kinematic differences exist between transtibial amputee fallers and non-fallers during downwards step transitioning.

Stair negotiation is biomechanically more challenging than level gait. There are few biomechanical assessments of transtibial amputees descending stairs and none specifically related to falls. Stair descent may elicit more differences than level gait in amputees with and without a previous falls history

Early Steps of HIV-1 Fusion Define the Sensitivity to Inhibitory Peptides That Block 6-Helix Bundle Formation

The HIV envelope (Env) glycoprotein mediates membrane fusion through sequential interactions with CD4 and coreceptors, followed by the refolding of the transmembrane gp41 subunit into the stable 6-helix bundle (6HB) conformation. Synthetic peptides derived from the gp41 C-terminal heptad repeat domain (C-peptides) potently inhibit fusion by binding to the gp41 pre-bundle intermediates and blocking their conversion into the 6HB. Our recent work revealed that HIV-1 enters cells by fusing with endosomes, but not with the plasma membrane. These studies also showed that, for the large part, gp41 pre-bundles progress toward 6HBs in endosomal compartments and are thus protected from external fusion inhibitors. Here, we examined the consequences of endocytic entry on the gp41 pre-bundle exposure and on the virus' sensitivity to C-peptides. The rates of CD4 and coreceptor binding, as well as the rate of productive receptor-mediated endocytosis, were measured by adding specific inhibitors of these steps at varied times of virus-cell incubation. Following the CD4 binding, CCR5-tropic viruses recruited a requisite number of coreceptors much faster than CXCR4-tropic viruses. The rate of subsequent uptake of ternary Env-CD4-coreceptor complexes did not correlate with the kinetics of coreceptor engagement. These measurements combined with kinetic analyses enabled the determination of the lifetime of pre-bundle intermediates on the cell surface. Overall, these lifetimes correlated with the inhibitory potency of C-peptides. On the other hand, the basal sensitivity to peptides varied considerably among diverse HIV-1 isolates and ranked similarly with their susceptibility to inactivation by soluble CD4. We conclude that both the longevity of gp41 intermediates and the extent of irreversible conformational changes in Env upon CD4 binding determine the antiviral potency of C-peptides

The role of agonist and antagonist muscles in explaining isometric knee extension torque variation with hip joint angle.

PURPOSE: The biarticular rectus femoris (RF), operating on the ascending limb of the force-length curve, produces more force at longer lengths. However, experimental studies consistently report higher knee extension torque when supine (longer RF length) compared to seated (shorter RF length). Incomplete activation in the supine position has been proposed as the reason for this discrepancy, but differences in antagonistic co-activation could also be responsible due to altered hamstrings length. We examined the role of agonist and antagonist muscles in explaining the isometric knee extension torque variation with changes in hip joint angle. METHOD: Maximum voluntary isometric knee extension torque (joint MVC) was recorded in seated and supine positions from nine healthy males (30.2 ± 7.7 years). Antagonistic torque was estimated using EMG and added to the respective joint MVC (corrected MVC). Submaximal tetanic stimulation quadriceps torque was also recorded. RESULT: Joint MVC was not different between supine (245 ± 71.8 Nm) and seated (241 ± 69.8 Nm) positions and neither was corrected MVC (257 ± 77.7 and 267 ± 87.0 Nm, respectively). Antagonistic torque was higher when seated (26 ± 20.4 Nm) than when supine (12 ± 7.4 Nm). Tetanic torque was higher when supine (111 ± 31.9 Nm) than when seated (99 ± 27.5 Nm). CONCLUSION: Antagonistic co-activation differences between hip positions do not account for the reduced MVC in the supine position. Rather, reduced voluntary knee extensor muscle activation in that position is the major reason for the lower MVC torque when RF is lengthened (hip extended). These findings can assist standardising muscle function assessment and improving musculoskeletal modelling applications

Copper Chaperone for Cu/Zn Superoxide Dismutase is a sensitive biomarker of mild copper deficiency induced by moderately high intakes of zinc

BACKGROUND: Small increases in zinc (Zn) consumption above recommended amounts have been shown to reduce copper (Cu) status in experimental animals and humans. Recently, we have reported that copper chaperone for Cu/Zn superoxide dismutase (CCS) protein level is increased in tissues of overtly Cu-deficient rats and proposed CCS as a novel biomarker of Cu status. METHODS: Weanling male Wistar rats were fed one of four diets normal in Cu and containing normal (30 mg Zn/kg diet) or moderately high (60, 120 or 240 mg Zn/kg diet) amounts of Zn for 5 weeks. To begin to examine the clinical relevance of CCS, we compared the sensitivity of CCS to mild Cu deficiency, induced by moderately high intakes of Zn, with conventional indices of Cu status. RESULTS: Liver and erythrocyte CCS expression was significantly (P < 0.05) increased in rats fed the Zn-60 and/or Zn-120 diet compared to rats fed normal levels of Zn (Zn-30). Erythrocyte CCS expression was the most sensitive measure of reduced Cu status and was able to detect a decrease in Cu nutriture in rats fed only twice the recommended amount of Zn. Liver, erythrocyte and white blood cell CCS expression showed a significant (P < 0.05) inverse correlation with plasma and liver Cu concentrations and caeruloplasmin activity. Unexpectedly, rats fed the highest level of Zn (Zn-240) showed overall better Cu status than rats fed a lower level of elevated Zn (Zn-120). Improved Cu status in these rats correlated with increased duodenal mRNA expression of several Zn-trafficking proteins (i.e. MT-1, ZnT-1, ZnT-2 and ZnT-4). CONCLUSION: Collectively, these data show that CCS is a sensitive measure of Zn-induced mild Cu deficiency and demonstrate a dose-dependent biphasic response for reduced Cu status by moderately high intakes of Zn