Count-dependent filter for smoothing bivariate FCM histograms

A data-smoothing filter has been developed that permits the improvement in accuracy of individual elements of a bivariate flow cytometry (FCM) histogram by making use of data from adjacent elements, a knowledge of the two-dimensional measurement system point spread function (PSF), and the local count density. For FCM data, the PSF is assumed to be a set of two-dimensional Gaussian functions with a constant coefficient of variation for each axis. A set of space variant smoothing kernels are developed from the basic PSF by adjusting the orthogonal standard deviations of each Gaussian smoothing kernel according to the local count density. This adjustment in kernel size matches the degree of smoothing to the local reliability of the data. When the count density is high, a small kernel is sufficient. When the density is low, however, a broader kernel should be used. The local count density is taken from a region defined by the measurement PSF. The smoothing algorithm permits the reduction in statistical fluctuations present in bivariate FCM histograms due to the low count densities often encountered in some elements. This reduction in high-frequency spatial noise aids in the visual interpretation of the data. Additionally, by making more efficient use of smaller samples, systematic errors due to system drift may be minimized.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Antigenic expression of B-cell chronic lymphocytic leukemic lymphocytes

A flow cytometric analysis of lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) samples and in monocyte-depleted and T-cell-depleted normal peripheral blood (B-PBL) samples was undertaken using 129 reagents from the blind panel (BP) and 72 reagents from the cluster designation (CD) panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B-Cell Section. After determining the average mean channel fluorescence and the average percentage of positive cells for the B-CLL and the normal B-PBL preparations, a combined ratio and difference analysis was performed for each monoclonal antibody reactivity. This analysis confirmed the intense expression of class II antigens on B-CLL and the preferential expression of CD19, CD20, CD23 and CD24 antigens. In addition, three new clustered and three new unclustered antigens were also preferentially expressed on B-CLL lymphocytes. Cluster analysis of these differences suggests the existence of at least three overlapping immunophenotypic subpopulations, composed of CD19, CD20, CD21, CD22, CD23, CD24, CD75, CD76 and CDw78.     

Biomechanical measurements on scaphoid bone screws in an experimental model

A number of screws commonly used for internal fixation in scaphoid bone fractures and nonunions are compared regarding biomechanical properties and clinical applicability. The experiments were carried out on models made of ash-wood, representing a reconstruction and fixation as is performed in a cortico-cancellous inlay bone graft for scaphoid non-union. For fixation use was made of 2.7 and 3.5 AO/ASIF cortical screws respectively, 4.0 AO/ASIF cancellous screws, Herbert screws, and a newly designed screw called the three components screw (D.K.S.). The models with implanted screws were tested for bending strength, tensile strength and torsion stability. No large differences between the various screws were found regarding the measured parameters, so that a small intra-osteal implant such as the Herbert screw and the D.K.S., which can be inserted easily and which gives a certain amount of interfragmentary compression, will be sufficient for osteosynthesis of the scaphoid bone. In case an intra-osteal implant is not available a single 3.5 AO/ASIF cortical screw, inserted following lag-screw principles, is recommended.     

Influence of neurophysin residues 1-8 on the optical activity of neurophysin-peptide complexes. Direct evidence that the 1-8 sequence alters the environment of bound peptide

Circular dichroism was used to compare the environment of peptides bound to native and des 1-8 neurophysin in order to further elucidate the role of the neurophysin 1-8 sequence in peptide-binding. A very large positive ellipticity (approximately 6000 deg cm2 dmol-1), shown earlier to be induced in tyrosine at position 2 of peptides bound to the native protein, was determined by the present study to be paralleled by similar induced changes in tyrosine at peptide position 1. Deletion of the neurophysin 1-8 sequence led to loss of half of the induced optical activity at peptide positions 1 and 2 and changes in binding-induced optical activity in the protein, the latter partially assignable to protein disulfides. In the mononitrated native and des 1-8 proteins, the optical activity of neurophysin Tyr-49, a residue at the peptide-binding site, was reduced by 80% in complexes of the des 1-8 protein relative to those of the native protein. The results suggest a role for neurophysin Arg-8 in modulating the optical activity at the binding site by directly placing a charge proximal to the binding site and/or by altering binding site conformation. The data provide the first unambiguous evidence of a difference in the environment of bound peptide between the native and des 1-8 proteins.     

Transfer of germ plasm from the secondary to the primary gene pool in pennisetum

Summary Germ plasm from the A-genome of Pennisetum purpureum Schum. (AABB) of the secondary gene pool was transferred to cultivated pearl millet (AA) [P. glaucum (L.) R. Br.] by pollinating cytoplasmicnuclear male-sterile (cms) pearl millet with fertile allohexaploid pearl millet x P. purpureum hybrids (AAAABB). Certain allohexaploids used as pollinators on cms pearl millet resulted in 14-chromosome diploid pearl millet progenies. Three types of diploid pearl millet plants were produced in addition to the expected 28-chromosome AAAB-genome plants: (1) cms plants with only the A-genome, (2) cms plants with the A- and A-genomes, and (3) fertile plants with the A- and A-genomes. The latter group has allowed the utilization of genes for fertility restoration, stiff stalk, maturity, height, and morphological characteristics from the A-genome of P. purpureum in the pearl millet breeding program. Production of monoploid gametes by the allohexaploids appeared to be genetically controlled.     

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.     

When are metal ion-dependent hydroxyl and alkoxyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide artifacts?

The formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/.OH adduct of the spin trap DMPO has been reported to occur through nucleophilic addition of water in the presence of aqueous ferric chloride (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990, Biochem. Biophys. Res. Commun. 172, 1073-1080). Due to the serious implications of these findings with respect to many spin trapping studies, the suitability of DMPO as a hydroxyl radical spin trap was studied in typical Fenton systems. Using 17O-enriched water, we show conclusively that nucleophilic addition of water occurs at the nitrone carbon (or C-2 position) of DMPO in the presence of either Fe or Cu ions. Furthermore, our results demonstrate that this nucleophilic reaction is a major pathway to the DMPO/.OH adduct, even during the reaction of Fe(II) or Cu(I) with hydrogen peroxide. Primary alkoxyl adducts of DMPO also form in aqueous solution through nucleophilic addition in the presence of both Fe(III) and Cu(II). Attempts to obtain secondary and tertiary alkoxyl adducts by this mechanism were unsuccessful, possibly due to steric effects. When the reaction is carried out in various buffers, however, or in the presence of metal ion chelators, nucleophilic addition to DMPO from Fe(III) is effectively suppressed. Chelators also suppress the reaction with Cu(II). Hence, under most common experimental conditions in biochemical free radical research, nucleophilic addition to DMPO should not be of major concern.