Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.     

N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness.

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.     

Physical mapping of the human carbonic anhydrase gene cluster on chromosome 8

A cluster of genes encoding the three cytoplasmic carbonic anhydrase isozymes CAI, CAII, and CAIII lie on the long arm of chromosome 8 (8q22) in humans. These genes have been mapped using pulsed-field gel electrophoresis. The genes lie in the order CA2, CA3, CA1. CA2 and CA3 are separated by 20 kb and are transcribed in the same direction, away from CA1. CA1 is separated from CA3 by over 80 kb and is transcribed in the direction opposite to CA2 and CA3. The arrangement of the genes is consistent with proposals that the duplication event which gave rise to CA1 predated the duplication which gave rise to CA2 and CA3. The order of these three genes differs from that suggested for the mouse based on recombination frequency.     

Polarity in higher plant dictyosomes

Summary Dictyosomes in three higher plant cell secretory systems,Zea root cap cells,Tradescantia pollen tubes and digestive glands ofDionaea fly traps, are shown to possess a structural polarity in terms of either the production of secretory vesicles, or the reaction of individual cisternae to the osmium-zinc-iodide impregnation procedure. These observations contradict previous claims that higher plant dictyosomes lack structural polarity. Doubts about the applicability of the endomembrane flow concept to higher plant cell dictyosomes are discussed in relation to the relative balance between carbohydrate and protein in secreted products. The lack of dictyosome-associated endoplasmic reticulum in some of these plant cell systems is confirmed.     

Histidine ammonia-lyase from Streptomyces griseus.

Histidine ammonia-lyase (histidase; HutH) has been purified to homogeneity from Streptomyces griseus and the N-terminal amino acid (aa) sequence used to clone the histidase-encoding structural gene, hutH. The purified enzyme shows typical saturation kinetics and is inhibited competitively by D-histidine and histidinol phosphate. High concentrations of K.cyanide inactivate HutH unless the enzyme is protected by the substrate or histidinol phosphate. On the basis of the nucleotide sequence, the hutH structural gene would encode a protein of 53 kDa with an N terminus identical to that determined for the purified enzyme. Immediately upstream from hutH is a region that strongly resembles a class of Streptomyces promoters active during vegetative growth; however, there is no obvious ribosome-binding site adjacent to the hutH translation start codon. The deduced aa sequence of an upstream partial open reading frame shows no similarity with other proteins, including HutP of Bacillus subtilis and HutU of Pseudomonas putida. Promoter-probe analysis indicates that promoter activity maps within the DNA surrounding the hutH start codon. Pairwise comparisons of the primary structures of bacterial and mammalian histidases, together with the unique kinetic properties and gene organization, suggest that streptomycete histidase may represent a distinct family of histidases.     

Multiple forms of vitamin D-dependent calcium-binding protein in rat kidney

The technique of two-dimensional electrophoresis was used in combination with a highly sensitive silver stain to study vitamin D-dependent calcium-binding protein (CaBP) in rat kidney. Rat renal CaBP was shown to co-migrate almost exactly with CaBP purified from chick intestine suggesting evolutionary conservation of this protein. In some cases rat renal CaBP appeared not as a single polypeptide, but rather as a cluster of 4 polypeptides. Formation of the satellite cluster of CaBP in response to high doses of 1,25-dihydroxyvitamin D3 occurred in young rats which had been maintained on a vitamin D-deficient diet for 2 weeks, as well as in older rats which had been maintained on the same diet for 5 months. The 4 forms of CaBP were not the result of various states of Ca2+ binding, but rather the result of an enzymatic reaction. This was shown by 3 experiments. 1) Adding excess EGTA to samples containing the 4 satellite forms did not change the two-dimensional electrophoretogram. 2) Incubation of purified chick intestinal CaBP with kidney cytosols from D-deficient rats brought about the formation of the satellite CaBP forms from the chick protein. However, purified chick CaBP was unchanged by incubation in buffer alone for up to 2 h at 37 degrees C. 3) Placing rat kidney cytosols in a boiling water bath for 10 min inactivated the factor which generated the satellite forms as would be expected for an enzyme. The physiological significance of these forms of CaBP is as yet unknown.     

The ability of nonspecific T-cell stimulators to induce helper-cell-dependent increases in either polyclonal or isotype-restricted Ig production in vivo

In order to delineate the various roles of T cells in B-cell activation, mice were exposed to a variety of specific or nonspecific T-cell stimuli including mitogens, e.g., concanavalin A, adjuvants, e.g., complete Freund's adjuvant, and colchicine plus nonimmunogenic doses of antigen, anti-lymphocyte serum, and pathogens and their spleens analyzed for total class-specific immunoglobulin-secreting cells as indicators of helper cell generation. The results demonstrate that, depending on the mode of stimulation, markedly different Ig-secreting cell response patterns were induced, differing with respect to their kinetics and the isotype induced. In contrast to polyclonal T-cell stimuli such as concanavalin A and 17X lethal malaria which induced increases in all classes of Ig-recruiting cells, injection of many T-cell-activating agents resulted in the selective production of IgG clones in particular IgG 1. Such findings are discussed in terms of the different mechanisms of T-cell help and provide further evidence for functional heterogeneity in the T-helper-cell pool.