Studies on the electrophoretic separability of B and T human lymphocytes.

Cell electrophoresis experiments were performed at physiological, ionic strength on Hypaque-Ficoll separated normal human peripheral blood lymphocytes. The initial lymphocyte preparations averaged 72% T cells and 22% B cells as estimated by resetting techniques. A bimodal electrophoretic distribution of fast T cells and slow B cells such as has been repeatedly shown to exist for experimental animals could not be demonstrated. Neither B- nor T-enriched populations showed any correlation between mobility and degree of enrichment. A strong positive correlation was found between the non-rosetting cells and a very slow electrophoretic subpopulation produced during the B enrichment procedure. It was also found that considerable variability in lymphocyte mobilities existed from person to person and even in the same individual sampled over several months; these observations imply that the pooling of electrophoretic data can be misleading. The importance of these findings lies in the growing interest in relating lymphocyte electrophoresis to disease processes and immunological rejection phenomena.     

Expression analysis of BACE2 in brain and peripheral tissues

Beta-site amyloid precursor protein cleaving enzyme (BACE) is a novel transmembrane aspartic protease that possesses all the known characteristics of the beta-secretase involved in Alzheimer's disease (Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. -C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741). We have analyzed the sequence and expression pattern of a BACE homolog termed BACE2. BACE and BACE2 are unique among aspartic proteases in that they possess a carboxyl-terminal extension with a predicted transmembrane region and together they define a new family. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA. In addition, in situ hybridization of adult rat brain shows that BACE2 mRNA is expressed at very low levels in most brain regions. The very low or undetectable levels of BACE2 mRNA in the brain are not consistent with the expression pattern predicted for beta-secretase.     

Discovery of orally available tetrahydroquinoline-based glucocorticoid receptor agonists

A series of tetrahydroquinoline derivatives were synthesized and profiled for their ability to act as glucocorticoid receptor selective modulators. Structure-activity relationships of the tetrahydroquinoline B-ring lead to the discovery of orally available GR-selective agonists with high in vivo activity.     

Nonsteroidal 2,3-dihydroquinoline glucocorticoid receptor agonists with reduced PEPCK activation

Continuing studies based on dihydroquinoline glucocorticoid receptor agonists lead to the discovery of a series of C4-oxime analogs. Representative compounds exhibited potent transrepression activity with minimal transactivation of phosphoenolpyruvate caboxykinase (PEPCK), a key protein in the gluconeogenesis pathway. These compounds represent promising leads in identifying GR agonists with high anti-inflammatory activity and attenuated potential for glucose elevation.     

PHYSICOCHEMICAL EFFECTS OF ALDEHYDES ON THE HUMAN ERYTHROCYTE

The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K⁺ leakage during fixation. The concentrations of K⁺ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.     

Identification of Neonatal White Matter on DTI: Influence of More Inclusive Thresholds for Atlas Segmentation

Purpose

Semi-automated diffusion tensor imaging (DTI) analysis of white matter (WM) microstructure offers a clinically feasible technique to assess neonatal brain development and provide early prognosis, but is limited by variable methods and insufficient evidence regarding optimal parameters. The purpose of this research was to investigate the influence of threshold values on semi-automated, atlas-based brain segmentation in very-low-birth-weight (VLBW) preterm infants at near-term age.

Materials and Methods

DTI scans were analyzed from 45 VLBW preterm neonates at near-term-age with no brain abnormalities evident on MRI. Brain regions were selected with a neonatal brain atlas and threshold values: trace <0.006 mm²/s, fractional anisotropy (FA)>0.15, FA>0.20, and FA>0.25. Relative regional volumes, FA, axial diffusivity (AD), and radial diffusivity (RD) were compared for twelve WM regions.

Results

Near-term brain regions demonstrated differential effects from segmentation with the three FA thresholds. Regional DTI values and volumes selected in the PLIC, CereP, and RLC varied the least with the application of different FA thresholds. Overall, application of higher FA thresholds significantly reduced brain region volume selected, increased variability, and resulted in higher FA and lower RD values. The lower threshold FA>0.15 selected 78±21% of original volumes segmented by the atlas, compared to 38±12% using threshold FA>0.25.

Conclusion

Results indicate substantial and differential effects of atlas-based DTI threshold parameters on regional volume and diffusion scalars. A lower, more inclusive FA threshold than typically applied for adults is suggested for consistent analysis of WM regions in neonates.     

Macrofiber structure and the dynamics of sickle cell hemoglobin crystallization

Fibers of deoxyhemoglobin S undergo spontaneous crystallization by a mechanism involving a variety of intermediate structures. These intermediate structures, in common with the fiber and crystal, consist of Wishner-Love double strands of hemoglobin S molecules arranged in different configurations. The structure of one of the key intermediates linking the fiber and crystal, called a macrofiber, has been studied by a variety of analytical procedures. The results of the analysis indicate that the intermediates involved in the fiber to crystal transition have many common structural features. Fourier analysis of electron micrographs of macrofibers confirms that they are composed of Wishner-Love double strands of hemoglobin molecules. Electron micrographs of macrofiber cross-sections reveal that the arrangement of the double strands in macrofibers resembles that seen in micrographs of the a axis projection of the crystal. This orientation provides an end-on view of the double strands which appear as paired dumb-bell-like masses. The structural detail becomes progressively less distinct towards the edge of the particle due to twisting of the double strands about the particle axis. Serial sections of macrofibers confirm that these particles do indeed rotate about their axes. The twist of the particle is right handed and its average pitch is 10,000 Å. The effect of rotation on the appearance of macrofiber cross-sections 300 to 400 Å thick can be simulated by a 15 ° rotation of an a axis crystal projection. The relative polarity of the double strands in macrofibers and crystals can be determined easily by direct inspection of the micrographs. In both macrofibers and crystals they are in an anti-parallel array.On the basis of these observations we conclude that crystallization of macrofibers involves untwisting and alignment of the double strands.