Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

The CLS2 gene encodes a protein with multiple membrane-spanning domains that is important Ca2+ tolerance in yeast

Genetic screening of Saccharomyces cerevisiae mutants defective in Ca²⁺ homeostasis identified cls2, which exhibits a specific Ca²⁺-sensitive growth phenotype. We describe here the CLS2 gene and a multicopy suppressor (named BCL21, for bypass of CLS2) of the cls2 mutation. The CLS2 gene encodes a polypeptide of 410 amino acid residues, and its hydropathy profile indicates that the predicted Cls2 protein (Cls2p) contains ten putative membrane spanning regions. Immunofluorescent staining of the yeast cells expressing epitopetagged Cls2p suggests that Cls2p is localized to endoplasmatic reticulum (ER) membrane. A cls2 disruption strain is viable, but shows a Ca²⁺-sensitive phenotype like the original cls2 mutants. BCL21 suppresses the cls2 disruption mutation, indicating that the multicopy suppression does not require the Cls2p. Suppression of cls2 was observed even after introduction of a singlecopy plasmid harboring BCL21. The BCL21 gene encodes a protein of 382 amino acid residues and is identical to the SUR1 gene. sur1 was originally isolated as a suppressor of rvs161, which has reduced viability in nutrient starvation conditions. Possible mechanisms of the multicopy suppression are discussed.     

Theste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.     

Mass propagation of shoots of Stevia rebaudiana using a large scale bioreactor

A procedure for the mass propagation of multiple shoots of Stevia rebaudiana is described. Isolated shoot primordia were used as the inoculum to obtain clusters of shoot primordia. Such clusters were grown in a 500 liter bioreactor to obtain shoots. A total of 64.6 Kg of shoots were propagated from 460 g of the inoculated shoot primordia. These shoots were easily acclimatized in soil.     

Possible Involvement of Abscisic Acid in Increases in Activities of Two Vacuolar H+-Pumps in Barley Roots under Aluminum Stress

Levels of abscisic acid (ABA) in barley roots increased upontreatment with AlCl₃. Treatment with AlCl₃ or ABA increasedboth ATP-dependent and PP_i-dependent H⁺-pumping activities intonoplast-enriched membrane vesicles. Increase in the H⁺-pumpingactivities caused by aluminum stress could result from increasedlevels of ABA. ¹Present address: Department of Botany, Faculty of Science,Hirosaki University, Hirosaki, Aomori, 036 Japan     

Fine genetic mapping of the proximal part of mouse Chromosome 2 excludes Pax-8 as a candidate gene for Danforth's short tail (Sd)

Danforth's short tail (Sd) is a semidominant mutation of the mouse with effects on the skeleton and the urogenital system. In view of its phenotype and its position in the proximal part of Chromosome (Chr) 2, three genes qualified as possible candidates: Pax-8, a paired box-containing gene; Midkine (Mdk), a retinoic acid-responsive gene; and a new locus (Etl-4) identified by enhancer trapping with a lacZ reporter gene which showed expression in the notochord, the mesonephric mesenchyme, and the apical ectodermal ridge. Three different backcrosses involving all three genes in different combinations were set up and analyzed. From our results we conclude that Sd, Etl-4, Pax-8, and Mdk are independent loci, with Etl-4 being the closest genetic marker (1.1±1.4 cM) to the Danforth's short tail (Sd) gene.     

Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture

The inhibitory effect of capsaicin on axoplasmic transport in cultured dorsal root ganglion cells was analyzed by video-enhanced contrast microscopy. Capsaicin inhibited particle transports in a dose-dependent manner, irrespective of the diameter of axons. The effect of capsaicin was reversible at low concentrations. Capsaicin affected both the anterograde and retrograde transport. Large organelles were more sensitive to capsaicin than small ones in the retrograde transport. An experiment using calcium-sensitive dye, Fura 2, indicated that capsaicin raised the intraneuronal free calcium concentration preceding the inhibition of the transport. Electron microscopy revealed that microtubules and neurofilaments are disorganized and disoriented by capsaicin. We reached a conclusion that capsaicin inhibits fast axoplasmic transport of both anterograde and retrograde directions in all types of somatosensory neurons in culture by disorganizing intraaxonal cytoskeletal structures, through the elevated intracellular Ca²⁺ concentration. © 1993 John Wiley & Sons, Inc.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.