Dual immunodetection and hybridisation in situ of opsin mRNA and rhodopsin protein in retinal sections

We describe a simple method for combining in situ hybridisation and immunohistochemistry on the same retinal section. The technique was developed using a radiolabelled cDNA probe for opsin and an antibody (ROS1F4) against rhodopsin. This method retains the antigenic sites if immunocytochemistry is performed prior to in situ hybridisation. Opsin mRNA was found in the photoreceptor inner segment with rhodopsin immunolocalised to the photoreceptor outer segments. The technique should be applicable to numerous situations including analysis of the sequence of events in the expression and synthesis of the various opsins during retinal development and degeneration.     

Fire evacuation skills: Cognitive-behavioral versus computer-mediated instruction

Fifty-six fourth-grade children participated in this study to assess three training procedures for relative effectiveness in acquiring fire emergency skills and attaining knowledge. The first training approach was behavioral, the second used animated graphics, and the third used still graphics. Each of the training groups was compared to a control group that received no treatment. Performance on dependent measures was assessed before and immediately after training. It was hypothesized that the behavioral and animated-graphics conditions would produce the greatest level of skill and knowledge acquisition. Results demonstrated a significantly higher level of fire emergency skill acquisition and knowledge attainment for both the behavioral and animated-graphics groups relative to the still-graphics group and the control group, which had no training. The value of using computer-mediated strategies for fire-safety skills training was demonstrated.     

Genetic fusions of subunit c in the F0 sector of H+-transporting ATP synthase. Functional dimers and trimers and determination of stoichiometry by cross-linking analysis

The multicopy c subunit of the H+-transporting ATP synthase of Escherichia coli folds through the transmembrane F0 sector as a hairpin of two hydrophobic alpha-helices with the proton-translocating aspartyl-61 side chain centered in the second transmembrane helix. The number of subunits c in the F0 complex, which is thought to determine the H+-pumping/ATP stoichiometry, was previously not determined with exactness but thought to range from 9-12. The studies described here indicate that the exact number is 12. Based upon the precedent of the subunit c in vacuolar-type ATPases, which are composed of four transmembrane helices and seem to have evolved by gene duplication of an F0-type progenitor gene, we constructed genetically fused dimers and trimers of E. coli subunit c. Both the dimeric and trimeric forms proved to be functional. These results indicate that the total number of subunit c in F0 should be a multiple of 2 and 3. Based upon a previous study in which the oligomeric organization of c subunits in F0 was determined by cross-linking of Cys-substituted subunits (Jones, P. C. , Jiang, W., and Fillingame, R. H. (1998) J. Biol. Chem. 273, 17178-17185), we introduced Cys into the first and last transmembrane helices of subunit c monomers, dimers, and trimers and attempted to generate cross-linked products by oxidation with Cu(II)-(1,10-phenanthroline)2. Double Cys substitutions at two sets of positions gave rise to extensive cross-linked multimers. Multimers of the monomer that extended up to the position of c12 were correlated and calibrated with distinct cross-linked species of the appropriate doubly Cys-substituted dimers (i.e. c2, c4, . c12) and doubly Cys-substituted trimers (i.e. c3, c6, c9, c12). The results show that there are 12 copies of subunit c per F0 in E. coli, the exact number having both mechanistic and structural significance.     

Weak interactions and lessons from crystallization

Ideas derived from the study of the process of crystallization may provide insights into molecular recognition in biological systems. Both processes exploit the cooperativity which arises from the formation of a large array of weak interactions.     

Phenotypic analysis of antigen-specific T lymphocytes

Identification and characterization of antigen-specific T lymphocytes during the course of an immune response is tedious and indirect. To address this problem, the peptide-major histocompatability complex (MHC) ligand for a given population of T cells was multimerized to make soluble peptide-MHC tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with two different human immunodeficiency virus (HIV)-derived peptides or with a peptide derived from influenza A matrix protein bound to peptide-specific cytotoxic T cells in vitro and to T cells from the blood of HIV-infected individuals. In general, tetramer binding correlated well with cytotoxicity assays. This approach should be useful in the analysis of T cells specific for infectious agents, tumors, and autoantigens.     

The gill calcium transport cycle in rainbow trout is correlated with plasma levels of bioactive, not immunoreactive, stanniocalcin

The aims of this study were to determine the most appropriate duration for the measurement of the maximal accumulated O2 deficit (MAOD), which is analogous to the anaerobic capacity, to ascertain the effects of mass, fat free mass (FFM), leg volume (Vleg) and lower body volume (V1b) on anaerobic test performance, to examine the reproducibility for peak power output (Wpeak) or maximal anaerobic power using an air-braked cycle ergometer and to produce approximations for the percentages of aerobic and anaerobic metabolism during exercise of short duration but high intensity. A group of 12 endurance trained cyclists [mean age 25.1 (SD 4.6) years; mean body mass 73.43 (SD 7.12) kg; mean maximal oxygen consumption 5.12 (SD 0.35) l.min-1; mean body fat 12.5 (SD 4.1) %] accordingly performed four counterbalanced treatments of 45, 60, 75 and 90 s of maximal cycling on an air-braked ergometer. The mean O2 deficit of 3.52 l for the 45-s treatment was significantly less (P < 0.01) than those for the 60 (3.75 l), 75 (3.80 l) and 90-s (3.75 l) treatments. These data therefore indicate that in predominantly aerobically trained subjects the O2 deficit attains a plateau after 60 s of maximal cycling on an air-braked ergometer. Statistically significant interclass correlation coefficients (P < 0.05) between the anthropometric variables (mass, FFM, Vleg and Vlb) and Wpeak or maximal anaerobic power (0.624-0.748) and MAOD (ml) or anaerobic capacity (0.666-0.772) furthermore would suggest the relevance of taking into account muscle mass during anaerobic tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

The electron microscopy of post-growth induced defects of ZnSe/GaAs epilayers

A fundamental research of structural defects induced upon post-growth processing of ZnSe/GaAs epilayers grown on (100) GaAs was done by identifying defect-related reflections in the transmission electron diffraction (TED) patterns of ZnSe. Structural artifacts, other than the as-grown defects, on this material system could be excluded according to our results. Four types of abnormal reflections have been observed in addition to primary reflections. These extra reflections are sensitive to the post-growth processing of ZnSe epilayers and may arise from various external effects, rather than epitaxy growth, such as irradiation damage, surface oxidation, and surface contamination. By mapping these reflections at several major zone axes using TED patterns, we found that the reciprocal lattice for a ZnSe crystal with structural defects consists of two distinct types of extra reflections associated with irradiation damage. The first type of extra reflections is ±1/3{111} and the other is ±1/2{111} corresponding to pure-edge and non-edge dislocation loops, respectively. For (100) oriented wafers, the ±1/3{111} and ±1/2{111} reflections were observed only on two of the four possible 〈111〉 variants (i.e. [111]_Zn and [111]_Zn)and this phenomenon was attributed to the anisotropy of defect distribution. Extra reflections associated with surface oxidation and contamination are also observed. The orientation relationships between a surface hexagonal ZnO and a cubic ZnSe film are [0001]_ZnO//[−111]_ZnSe, and [01−11]_ZnO//[011]_ZnSe. The origin, characterization, and elimination of these induced reflections are discussed. With the knowledge about these extra effects on structural defect formation, we have shown the real microstructure of ZnSe epilayers.