Expression of monoclonal antibody-defined cell surface antigens during rat brain development

Using single-cell suspensions of mechanically dissociated, prenatal BDIX-rat brain cells (13th, 15th, and 21st days after fertilization) for immunization, we have established a collection of 37 monoclonal antibodies (Mabs) directed against neural cell surface determinants. The developmental-stage-dependent expression of cell-surface antigens recognized by these Mabs was analyzed both on plasma membranes isolated from whole brains of BDIX rats (prenatal days 13-22 and adults) using an indirect 125I solid-phase radioimmunoassay, and on intact BDIX-rat brain cells (prenatal days 13-22) using a fluorescence-activated cell sorter. Different types of developmental stage-dependent profiles of Mab binding were found, these being indicative of the presence of neural cell surface determinants whose expression increases, decreases, or does not change with brain development. Some of the Mab-binding profiles showed transient changes as a function of developmental stage. These Mabs are currently being used for the characterization, reproducible identification, and isolation of neural cell subpopulations of the developing rat brain, with the aim of investigating the cell type dependence and developmental (differentiation) stage dependence of malignant transformation following pulse exposure to the carcinogen N-ethyl-N-nitrosourea at defined stages of brain development.     

Contitions for optimal growth of a PSTV-infected potato cell suspension and detection of viroid-complementary longer-than-unit-length RNA in these cells

A suspension culture from potato spindle tuber viroid (PSTV)-infected cells of the wild type potato (Solanum demissum) has been established, which is a suitable model system for studying PSTV replicationin vivo. The conditions for rapid growth of these cells and for permanent extensive viroid biosynthesis within them are described. Biosynthesis of PSTV in the potato cells was demonstrated by³²P-incorporation into nucleic acids and their subsequent electrophoretic analysis on polyacrylamide gels. Under optimum culture conditions the amount of³²P-orthophosphate incorporation into PSTV reached 10% of that incorporated into the 2 M LiCl-soluble cellular RNA. (+)PSTV and its complementary form, i.e. (?)PSTV were identified after their electrophoretic separation on polyacrylamide and agarose gels by molecular hybridization. This analysis revealed the presence of six high molecular weight(?)PSTV species, which are possibly multimers of the unit length(+)PSTV molecule consisting of 359 nucleotides.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

The Urokinase Receptor Is a Major Vitronectin-Binding Protein on Endothelial Cells

We have previously demonstrated that vitronectin (VN), a morphoregulatory protein in the vessel wall, is internalized and translocated to the subendothelial matrix by an integrin-independent mechanism (J. Histochem. Cytochem.41, 1823–1832, 1993). The cell surface component which mediates the initial contact of VN with endothelial cells is defined here. The specific binding of VN to endothelial cells demonstrated the following properties: a threefold increase after phorbol ester treatment; 85% inhibition by pretreatment of cells with phosphatidylinositol–phospholipase C to release glycolipid-anchored surface proteins; a 90% inhibition by urokinase (u-PA) receptor blocking antibody. u-PA increased VN binding to cells due to an eightfold increase in the affinity of VN for the u-PA receptor. Structure–function studies showed that the amino-terminal fragment of u-PA, devoid of any proteolytic activity, mediated this effect. Active plasminogen activator inhibitor-1 (PAI-1), but not inactivated PAI-1, inhibited VN binding to cells and displaced VN that was prebound to endothelial cell monolayers. Similarly, VN binding to purified (immobilized) u-PA receptor, but not to integrin, was enhanced by u-PA and inhibited by PAI-1. Hence, the binding of soluble VN to endothelial cell surfaces is mediated by the u-PA receptor, and the relative concentrations of u-PA and PAI-1 are able to regulate the strength of this interaction. Endothelial cell adhesion to immobilized VN was found to be integrin-mediated without any involvement of the VN–uPA-receptor system. Hence, the interaction of VN with the u-PA receptor may be involved in the regulation of cellular processes necessary for endothelial cell invasion and migration at VN-rich extracellular matrix sites.     

The coupling of crossing over and homologous synapsis in maize inversions

Because fresh initiations of synapsis must occur for homologous synapsis of internal heterozygously inverted chromosome segments, attention has been directed at homologous synapsis and crossing over in overlapping paracentric inversions in the long arm of chromosome 1 of maize. In an earlier study with a relatively short inversion (where double crossovers within the inversion were rare), a recombination nodule (RN) was generally found at pachytene in reverse paired (homologously synapsed) inverted regions. Crossover frequency within the inversion, which could be independently estimated from analysis of bridge and fragment frequency at anaphase I and II, closely corresponded to crossover frequency estimated from observed RN frequency in pachytene inversion loops. These findings were consistent with the interpretation that establishment of homologous synapsis in this case is generally coupled to crossing over. This coupling suggests that there is very early commitment to the form of resolution of recombination intermediates that results in reciprocal recombination events instead of conversion only or other noncrossover events. This study examines another, larger paracentric inversion in the long arm of chromosome 1 that completely overlaps the first inversion. It is sufficiently longer than the first inversion that double crossover events are found within it with substantial frequency and interference considerations are feasible. This study confers additional insight into the interrelationships of synapsis and crossing over and the probable sequence in which the various involved processes usually occur. It raises the strong possibility that crossovers can be initiated during the alignment phase that precedes synapsis.     

Synaptic defects of asynaptic homozygotes in maize at the electron microscope level.

More detailed observations of the synaptonemal complex (SC) in asynaptic maize plants have been faciliated by superior silver-staining procedures. These suggest that central region components of the SC are strongly implicated as defective in asynaptic. Apparently homologous axial elements tend to follow roughly parallel courses within the nucleus at pachytene, in some short segments apparently synapsed and in others at wider separation than normal synapsis yet close enough to allow observation of thin central element segments and also occasional thin transverse element-type structures. This kind of transverse filament may be weakened and severely stretched yet associated with both axial elements. Small nodules, similar to recombination nodules, appear at corresponding positions in widely separated axial elements. Key words : synaptonemal complex, central element, transverse filament, recombination nodule.     

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S 1 during the transition. Correspondence to: Y. K. Levine     

Pool sequencing of natural HLA-DR,DQ, and DP ligands reveals detailed peptide motifs,constraints of processing,and general rules

We have approached the problem of MHC class II ligand motifs by pool sequencing natural peptides eluted from HLA-DR, DQ, and DP molecules. The results indicate surprisingly clear patterns, although not quite as clear as with natural class I ligands. The most striking feature is a highly dominant Proline at position 2. We interpret this to be a consequence of aminopeptidase N-like activity in processing. Another general aspect is the existence of three to four hydrophobic or aromatic anchors, whereby the first and the last are separated by five to eight residues. The peptide motifs for HLA-DR1, DR5, DQ7, and DPw4 are allele-specific and differ by spacing and occupancy of anchors. The anchors tend to be flanked by clusters of charged residues, and small residues, especially Ala, are frequent in the motif centers. These detailed motifs allow one to interpret most previous (DR-) motifs as fitting one or more of the anchors or conserved clusters. The relative motif symmetry suggests the possibility of bidirectional binding of peptides in the class II groove.