Immunohistochemical co-localization of distinct connexins (Cxs) in junctional areas suggests the formation of heteromultimeric channels. To determine the docking effects of the heterotypic ...combination of Cx43 and Cx45 on the voltage-gating properties of their channels, we transfected DNA encoding Cx43 or Cx45 into N2A neuroblastoma or HeLa cells. Using a double whole-cell voltage-clamp technique, we determined macroscopic and single-channel gating properties of the intercellular channels formed. Cx43-Cx45 heterotypic channels had rectifying properties where Cx45 connexons inactivated rapidly upon hyperpolarizing voltage pulses applied to the Cx45-expressing cell. During depolarizing pulses to the Cx45-expressing cell, Cx43 connexons inactivated with substantially reduced kinetics as compared with homotypic Cx43 channels. Similar slow kinetics was observed for homotypic Cx43M257 (truncation mutant). Heterotypic channels had a main conductance whose value was predicted by the sum of corresponding homomeric connexon conductances; it was not voltage dependent and had no detectable residual conductance. The voltage-gating kinetics of heterotypic channels and their single-channel behavior implicate a role for the Cx43 carboxyl-terminal domain in the fast gating mechanism and in the establishment of residual conductance. Our results also suggest that heterotypic docking may lead to conformational changes that inhibit this action of the Cx43 carboxyl-terminal domain.
Although current influenza vaccines have been shown to reduce influenza-related morbidity and mortality, there is a desire to develop more efficacious products. Vaccines which can induce CD8
+ ...cytotoxic T cell (CTL) responses in addition to strong antibody responses may be more effective in preventing disease since it has been demonstrated that CTL contribute to protective immunity, even against drift variants of influenza A viruses. The immunogenicity of two types of experimental influenza vaccines, which were based on immune stimulating complexes (ISCOM™), were evaluated and compared with a conventional non-adjuvanted inactivated split virion vaccine, after immunization of human volunteers. In this randomized, double blind study, it was shown that the ISCOM™ vaccines altered the kinetics of the serum antibody response, resulting in more rapid titer rises against the vaccine strains. This accelerated antibody response coincided with enhanced in vitro proliferative T cell responses, which were observed shortly after vaccination. In addition, CTL responses were observed in a higher proportion of the vaccinees receiving an ISCOM™ vaccine, than in vaccinees receiving the conventional influenza vaccine.
•A N2 ice glacial flow model is developed.•The model incorporates known thermophysical and rheological properties of N2.•Dark patterning seen near Sputnik Planitia’s northern shoreline is ...examined.•Our flow model suggests these may be imprints of near surface bottom topography.
Observations of Pluto’s surface made by the New Horizons spacecraft indicate present-day N2 ice glaciation in and around the basin informally known as Sputnik Planitia. Motivated by these observations, we have developed an evolutionary glacial flow model of solid N2 ice that takes into account its published thermophysical and rheological properties. This model assumes that glacial ice flows laminarly and has a low aspect ratio which permits a vertically integrated mathematical formulation. We assess the conditions for the validity of laminar N2 ice motion by revisiting the problem of the onset of solid-state buoyant convection of N2 ice for a variety of bottom thermal boundary conditions. Subject to uncertainties in N2 ice rheology, N2 ice layers are estimated to flow laminarly for thicknesses less than 400–1000 m. The resulting mass-flux formulation for when the N2 ice flows as a laminar dry glacier is characterized by an Arrhenius–Glen functional form. The flow model developed is used here to qualitatively answer some questions motivated by features we interpret to be a result of glacial flow found on Sputnik Planitia. We find that the wavy transverse dark features found along the northern shoreline of Sputnik Planitia may be a transitory imprint of shallow topography just beneath the ice surface suggesting the possibility that a major shoreward flow event happened relatively recently, within the last few hundred years. Model results also support the interpretation that the prominent darkened features resembling flow lobes observed along the eastern shoreline of the Sputnik Planitia basin may be the result of a basally wet N2 glacier flowing into the basin from the pitted highlands of eastern Tombaugh Regio.
The results of the haemagglutination-inhibiting (HI) antibody test for influenza virus antibody in human sera closely match those produced by virus neutralization assays and are predictive of ...protection. On the basis of the data derived from 12 publications concerning healthy adults, we estimated the median HI titre protecting 50% of the vaccinees against the virus concerned at 28. This finding supports the current policy requiring vaccines to induce serum HI titres of > or = 40 to the vaccine viruses in the majority of the vaccinees. Unfortunately similar studies are scanty for the elderly, the group most at risk of influenza. There still remain many unsolved technical problems with the HI assay and we recommend that these problems be studied and the virus neutralization test as a predictor of resistance to influenza be assessed. Although the studies on this issue often give conflicting results, they generally show that HI antibody responses to influenza vaccination tend to diminish with increasing age, when health is often compromized. Advanced age in itself seems not to be an independent factor in this process. However, even in completely healthy elderly individuals the response to vaccination with an antigenically new virus may be strongly reduced compared with younger vaccinees.
CD33 is expressed by acute myeloid leukemia (AML) cells in >80% of patients but not by normal hematopoietic stem cells, suggesting that elimination of CD33+ cells may be therapeutically beneficial. A ...conjugate of a calicheamicin hydrazide derivative attached via hydrazone formation to the oxidized carbohydrates of the anti-CD33 murine antibody P67.6 had been chosen for use in AML prior to humanization of this antibody. However, the CDR-grafted humanized P67.6 could not be used to make the carbohydrate conjugate because of the unexpected sensitivity of this antibody to periodate oxidation. Exploration of a series of bifunctional linkers resulted in a new class of calicheamicin conjugates, termed the hybrid conjugates, that allows for the attachment of the calicheamicin to lysines but incorporates the site of hydrolytic release, a hydrazone, previously shown to be required for activity. The optimized conjugate chosen for clinical trials, gemtuzumab ozogamicin (“gem-ozo”, Mylotarg, formerly designated CMA-676), was significantly more potent and selective than the carbohydrate conjugate it replaced. It was selectively cytotoxic to HL-60 leukemia cells in tissue culture with an IC50 in the low to sub-pg cal/mL range (cal = calicheamicin equivalents). Doses of gem-ozo as low as 50 μg cal/kg given three times to mice bearing HL-60 xenografts routinely resulted in long-term, tumor-free survivors, while a nonbinding control conjugate was relatively inactive. Gem-ozo at a concentration of 2 to 10 ng cal/mL selectively inhibited leukemia colony formation by marrow cells from a significant proportion of AML patients. Gem-ozo has also shown significant activity against AML in Phase II trials and is the first antibody-targeted chemotherapeutic agent approved by the FDA.
Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0-kb band. This band was observed only after hybridization ...and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5′ end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.
Two 18-d studies were conducted to evaluate the effects of drought stress and carbohydrase enzyme inclusion on broiler performance and nutrient digestibility in either yellow dent corn or sorghum. ...Both experiments employed a 2 × 2 factorial arrangement of treatments with 2 growing environments (normal or stressed), and carbohydrase enzyme cocktail (yes or no). In each experiment, 256 chicks were randomly assigned with 8 birds/pen and 8 replicates/treatment. Enzyme inclusion did not affect any variable in either experiment, and environment did not alter 18-d BW, mortality, or the digestibility of DM or CP. In Experiment 1, birds consuming normal corn had improved FCR compared to those fed drought-affected corn (1.27 vs. 1.33, respectively), which was potentially driven by the difference in apparent total tract digestibility (ATTD) of EE. Alternatively, FCR was not affected by environment in birds fed sorghum-based diets in Experiment 2, but those that consumed normal sorghum had lower feed intake than those fed drought-affected corn (750 vs. 796 g, respectively). In summary, commercial carbohydrase inclusion did not improve growth performance or nutrient digestibility in birds fed normal or drought-affected corn or sorghum. Birds fed drought-affected corn and sorghum affected growth performance variables, but environment did not affect final weight or mortality of broiler chicks.
Connexins form a variety of gap junction channels that vary in their developmental and tissue-specific levels of expression, modulation of gating by transjunctional voltage and posttranslational ...modification, and unitary channel conductance (gamma sub j). Despite a 10-fold variation in gammaj, whether connexinspecific channels possess distinct ionic and molecular permeabilities is presently unknown. A major assumption of the conventional model for a gap junction channel pore is that gamma sub j is determined primarily by pore diameter. Hence, molecular size permeability limits should increase and ionic selectivity should decrease with increasing channel gammaj (and pore diameter). Equimolar ion substitution of 120 mmol/L KCl for potassium glutamate was used to determine the unitary conductance ratios for rat connexin40 and connexin43, chicken connexin43 and connexin45, and human connexin37 channels functionally expressed in communication-deficient mouse neuroblastoma (N2A) cells. Comparison of experimental and predicted conductance ratios based on the aqueous mobilities of all ions according to the Goldman-Hodgkin-Katz current equation was used to determine relative anion-to-cation permeability ratios. Direct correlation of junctional conductance with dye transfer of two fluorescein-derivatives (2 mmol/L 6-carboxyfluorescein or 2 prime,7 prime-dichlorofluorescein) was also performed. Both approaches revealed a range of selectivities and permeabilities for all five different connexins that was independent of channel conductance. These results are not consistent with the conventional simple aqueous pore model of a gap junction channel and suggest a new model for connexin channel conductance and permselectivity based on electrostatic interactions. Divergent conductance and permeability properties are features of other classes of ion channels (eg, Na and K channels), implying similar mechanisms for selectivity.(Circ Res. 1995;77:1156-1165.)
We built and measured radio-frequency (RF) loss tangent, tan δ, evaluation structures using float-zone quality silicon-on-insulator (SOI) wafers with 5 μm thick device layers. Superconducting Nb ...components were fabricated on both sides of the SOI Si device layer. Our main goals were to develop a robust fabrication for using crystalline Si (c-Si) dielectric layers with superconducting Nb components in a wafer bonding process and to confirm that tan δ with c-Si dielectric layers was reduced at RF frequencies compared to devices fabricated with amorphous dielectrics, such as SiO2 and SixNy, where tan δ ∼ 10-3. Our primary test structure used a Nb coplanar waveguide (CPW) readout structure capacitively coupled to LC resonators, where the capacitors were defined as parallel-plate capacitors on both sides of a c-Si device layer using a wafer bonding process with benzocyclobutene (BCB) wafer bonding adhesive. Our control experiment, to determine the intrinsic tan δ in the SOI device layer without wafer bonding, also used Nb CPW readout coupled to LC resonators; however, the parallel-plate capacitors were fabricated on both sides of the Si device layer using a deep reactive ion etch (DRIE) to access the c-Si underside through the buried oxide and handle Si layers in the SOI wafers. We found that our wafer bonded devices demonstrated F· δ = (8 ± 2) × 10-5, where F is the filling fraction of two-level states (TLS). For the control experiment, F· δ = (2.0 ± 0.6) × 10-5, and we discuss what may be degrading the performance in the wafer bonded devices as compared to the control devices.
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