A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.     

20-Hydroxyecdysone mediated activation of larval haemolymph protein uptake by fat body cells of Corcyra cephalonica (Insecta)

Evidence is presented here to show that 20-hydroxyecdysone is essential for the activation of the larval fat body for differential uptake of larval haemolymph proteins (LHPs). By using radiolabelled LHPs it is shown that the fat body cells of Corcyra cephalonica selectively incorporate LHPs during late-larval and prepupal development. Fluorographic analysis of the labelled fat body proteins from prepupal stage separated on sodium dodecyl-sulphate polyacrylamide gels suggests that the LHPs are sequestered without any degradation. Although, during the last larval instar the uptake of all the three LHPs (LHP 1, LHP 2 and LHP 3) by the fat body cells is very low, 20-hydroxyecdysone treatment of early, mid or late-last instars causes a significant increase in uptake of all the three LHPs. However, the response to hormone treatment was more pronounced in late-last instar when compared to early and mid-last instar.     

Changes in erythrocyte glutathione peroxidase and glutathione reductase in alloxan diabetes

Glutathione peroxidase and glutathione reductase activities were measured in erythrocytes from control, diabetic and insulin-treated diabetic rats. A significant decrease in the activity of glutathione peroxidase and an increase in the glutathione reductase activity were found with increase in the time of diabetes which may result in the alteration in the activity of the pentose phosphate pathway by the modulation of the levels of NADPH. Insulin administration reverses the change in the activity of glutathione peroxidase but does not reverse the glutathione reductase activity during diabetes. The overall changes may be due to changes in the levels of insulin, triiodothyronine and thyroxine.     

Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice

Rice long repetitive DNA (9–20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Cam^r Amp^r Tet^s), only a few were selected randomly for further characterization. The insert size in all these clones was 3–4 kbp. Restriction enzyme analysis showed the absence ofEcoRI andBclI sites, presence of a singlePstI andPvuII site and multiple sites forAluI in 3 clones namely pRLl, pRL7 and pRL10. TheBamHI-PstI fragment of about 0.4 kbp in the pRL7 insert DNA (pRL7-0.4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25 S rDNA sequence of rice, however, hybridisation did not indicate any homology.     

Horse heart ferricytochromec: Conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochromec in thepH range 6–0.8 and at the lowest possible ionic strengths, in water, and at 30°C are reported. The neutralpH form, state III, changes to the acidicpH form, state I, through a three-step process: state III ? state IIIa ? state II ? state I, with pKa's of 3.6±0.3, 2.7±0.2, and 1.2±0.2, depending on the monitoring probe, respectively. State IIIa ferricytochromec is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochromec is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 Å.     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.