I-Region genetic restrictions imposed upon the recognition of KLH by murine T-cell clones

Data presented in this paper show that the recognition of keyhole limpet hemocyanin by murine T-cell clones is restricted by products of the I region. These data have been obtained by genetic mapping studies as well as by the use of monoclonal la-specific antibodies which inhibit the ability of antigen-presenting cells to effectively present antigen to such T-cell clones. Use of heterozygous antigen-presenting cells derived from crosses between B6.C-H-2 ^bm12 and B10.A(4R) mice have allowed us to show that both trans-complementing I-A products are used for restriction of recognition of KLH. These data were confirmed using monoclonal Ia antibodies to inhibition recognition of KLH by the same T-cell clones. Thus, we have shown that there exist hybrid molecules formed by free combinatorial association of products encoded within the I-A subregion which restrict the recognition of soluble antigen. Additionally, we have shown that the molecule formed by complementation between the alpha chain encoded within the I-E region and a beta chain encoded within the A region (A_e) can function effectively in presenting KLH to certain murine T-cell clones. These results support the hypothesis that the recognition of individual antigenic epitopes within large multideterminant antigens is under the control of Ir genes.     

Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization.

The identification of differential gene expressionbetween cells is a frequent goal in modern biological research. Here we demonstrate the coupling of representational difference analysis (RDA) of cDNA with microarray analysis of the output for high throughput screening. Two primary Ewing's sarcoma tissue samples with different biological behavior in vivo were compared by RDA: one which was metastatic and progressed rapidly; the other localized and successfully treated. A modified RDA protocol that minimizes the necessary starting material was employed. After a reduced number of subtractive rounds, the output of RDA was shotgun cloned into a plasmid vector. Inserts from individual colonies from the subtracted library were amplified with vector-specific primers and arrayed at high density on glass slides. The arrays were then hybridized with differentially fluorescently labeled starting amplicons from the two tissues and fluorescent signals were measured at each DNA spot. We show that the relative amounts of fluorescent signal correlate well with the abundance of fragments in the RDA amplicon and in the starting mRNA. In our system, we analyzed 192 products and 173 (90%) were appropriately detected as being >2-fold differentially expressed. Fifty unique, differentially expressed clones were identified. Therefore, the use of RDA essentially provides an enriched library of differentially expressed genes, while analysis of this library with microarrays allows rapid and reproducible screening of thousands of DNA molecules simultaneously. The coupling of these two techniques in this system resulted in a large pool of differentially expressed genes.     

Electron microscopy of the transitional conversion cell of Histoplasma capsulatum

Aspects of the fine structure of the transitional conversion cell formed during the early stages of the yeast to mold morphogenesis ofHistoplasma capsulatum as seen in ultrathin sections are described and illustrated by electron micrographs. Formation of the transitional cell was observed to occur with the highest degree of frequency between the 18th and 24th hr following induction of the conversional stimulus, although many yeastlike cells were observed to undergo degeneration or to initiate conversion only to abort the process. Cytoplasmic streaming and organelle migration from the parent yeast to the transitional cell was observed to occur prior to septation. The cell wall of the transitional form is thinner than that of the yeast and appears to arise from the inner portion of the laminated cell wall adjacent to the plasma membrane of the converting yeastlike cell. Interseptal or Woronin bodies were observed in association with the septal pore of the completed septum and were observed in the cytoplasm of both the yeastlike and transitional cell. The presence of these structures support strongly the pre-hyphal character of the converting cell complex.     

Scanning-beam electron microscopy of the conidia of the brown and albino filamentous varieties of Histoplasma capsulatum

Aspects of the surface appearance and external morphology of the conidial forms of the albino and brown filamentous varieties ofHistoplasma capsulatum as seen by scanning-beam electron microscopy are described and illustrated by electron micrographs. Septal areas between the hyphal cells of the supporting mycelium are seen as slightly elevated annular rings or ridges. The smooth micro- and macroconidia of the albino filamentous variety show a fine wrinkling and delicate irregularities of surface texture. Macroconidia of the brown filamentous variety are illustrated showing variations in numbers and respective length of the conspicuous wall projections or tubercles. The 3-dimensional perspective, unusual depth of focus, and high resolving power of the stereoscan technique permitted observations of external conidia morphology unattainable by other methods of study.     

STUDIES IN SHOOT-TIP ABORTION: SYRINGA VULGARIS

Garrison , Rhoda (Wheaton Coll., Norton, Mass.), and Ralph H. Wetmore . Studies in shoot-tip abortion: Syringa vulgaris. Amer. Jour. Bot. 48(9): 789–795. Illus. 1961.—The growth of shoots of Syringa vulgaris is described with special reference to the sequence of events in the abortion of the tip. Early in the growing season, as the shoot begins to elongate, the terminal part, 4–7 mm in length and bearing 3 or 4 pairs of young leaves, ceases growth; then after 2 or 3 wk in which there is no change in form, the tip becomes yellow and disintegrates. Diffusible auxin is present in the terminal region as a shoot begins to elongate, but after the abortive phase is initiated, auxin can no longer be detected in that part. Young shoots and tips of shoots when cultured in nutrient media ultimately abort in the normal manner. Shoot tips in the early stages of abortion can be stimulated to resume growth and development if the axillary buds just subjacent to the tip are destroyed; this response is, however, limited to vigorously growing shoots.     

Development of an Assay for Phospholipase C Using Column-Reconstituted, Extruded Phospholipid Vesicles

The reconstitution of heterotrimeric G proteins into phospholipid vesicles has been widely used for the measurement of PLC-beta activity in vitro. We have developed an improved and sensitive method for the assay of PLC-beta activity. This approach involves reconstitution of purified betagamma dimers into extruded phospholipid vesicles containing phosphatidylinositol 4, 5-bisphosphate and using a gel-filtration technique to separate the reconstituted vesicles from monodispersed betagamma dimers and the detergent used to solubilize G proteins. The method provides physical information about the partitioning of betagamma dimers into phospholipid vesicles and was used to examine the effect of different prenyl groups on the gamma subunits in the activation of PLC-beta. The beta1gamma1 dimer (containing the farnesyl group) and the beta1gamma2 dimer (containing the geranylgeranyl group) were purified from baculovirus-infected Sf9 insect cells and were found to partition equally into phospholipid vesicles. The beta1gamma2 dimer is more potent and effective in stimulating PLC-beta activity than the beta1gamma1 dimer. The EC50 values of betagamma dimers for the activation of PLC-beta determined with this method were lower than those determined by previous methodology, showing that betagamma subunits have a subnanomolar affinity for PLC-beta.