Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

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Long promoter sequences form higher-order G-quadruplexes: an integrative structural biology study of c-Myc,k-Ras and c-Kit promoter sequences

We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34–70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.     

Phenotype and Hierarchy of Two Transgenic T Cell Lines Targeting the Respiratory Syncytial Virus KdM282-90 Epitope Is Transfer Dose-Dependent

In this study, we compared two lines of transgenic CD8+ T cells specific for the same K^dM2_82-90 epitope of respiratory syncytial virus in the CB6F1 hybrid mouse model. Here we found that these two transgenic lines had similar in vivo abilities to control viral load after respiratory syncytial virus infection using adoptive transfer. Transfer of the TRBV13-2 line resulted in higher levels of IL-6 and MIP1-α in the lung than TRBV13-1 transfer. Interestingly, when large numbers of cells were co-transferred, the lines formed a hierarchy, with TRBV13-2 being immunodominant over TRBV13-1 in the mediastinal lymph node despite no identifiable difference in proliferation or apoptosis between the lines. This hierarchy was not established when lower cell numbers were transferred. The phenotype and frequency of proliferating cells were also cell transfer dose-dependent with higher percentages of CD127^loCD62L^loKLRG1^lo and proliferating cells present when lower numbers of cells were transferred. These results illustrate the importance of cell number in adoptive transfer experiments and its influence on the phenotype and hierarchy of the subsequent T cell response.     

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

Intestinal lactase in the neonatal rat. Maturational changes in intracellular processing and brush-border degradation

The mechanism of decline in the catalytic activity of intestinal lactase during neonatal maturation has not been defined, but a shift in the lactase subunit synthesis from an active 130-kDa subunit to an inactive 100-kDa species has now been noted in the adult rat (Quan, R., Santiago, N. A., Tsuboi, K. K., and Gray, G. M. (1990) J. Biol. Chem. 265, 15882-15888). The subunit structure, synthesis, intracellular assembly, and subsequent degradation of lactase from the brush-border surface membrane was examined in 15-day-old pre-weaned and 30-day-old post-weaned intact rats. Lactase was labeled intraintestinally with [35S]methionine, isolated from Triton-solubilized membranes with monospecific polyclonal anti-lactase, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The protein-stained gel revealed subunits of 225 and 130 kDa, the latter species predominating in both the pre- and post-weaned state. The distinct adult-type 100-kDa moiety was present in post-weaned animals while only a trace of a slightly larger (approximately 110 kDa) species was observed in pre-weaned animals. Quantitation of radioactivity in newly synthesized lactase revealed an increasing prominence of the 100-kDa species in post-weaned rats (130/100 incorporation ratio: pre-weaned 6.2; post-weaned 3.3). Accumulation of newly labeled lactase in brush-border membranes after intraperitoneal [35S]methionine labeling was similar in both groups at 3 h. Despite these comparable rates of lactase synthesis, assembly and insertion in the pre- and post-weaned state, subsequent removal of the 130-kDa unit was more rapid in post-weaned animals (t1/2 = 11 h; pre-weaned t1/2 = 37 h). In intact rats, the neonatal maturational decline in lactase catalytic activities involves both a shift to production of the inactive 100-kDa subunit and increased membrane surface degradation of the active 130-kDa subunit.     

A Rocking Table for Staining Electrophoretic Gels, Nitrocellulose Blots and Histological Slides

A rocking table has been designed for gentle cyclic agitation of electrophoretic gels, nitrocellulose blots and microscope slides during staining (Fig. 1). It is particularly useful for long staining periods or when a small volume of staining solution is to be used. Even under conditions where gels or slides, laid flat, are not fully submerged when the staining vessel is horizontal, staining is consistently even and satisfactory. The apparatus is simple and inexpensive to construct, easily operated and quiet during use. It can be operated over a temperature range from —20 to +60 C. The rocking speed is continuously variable from 4 to 14 cycles per minute. This allows precise control to avoid gross disturbance of the materials being stained, or splashing of the stain solution in different containers.