The glycosylation pattern of a humanized IgGl antibody (D1.3) expressed in CHO cells

A humanized IgG antibody (D1.3) which retains murine complementarity determining regions specific for the antigen lysozyme has been expressed in CHO-DUKX cells. Heavy and light chain containing plasmids were co-transfected into CHO-DUKX cells and stable clones were grown in DMEM/F12 medium supplemented with 5% foetal calf serum. D1.3 antibody was purified from culture supernatants by Protein G chromatography. With the recombinant D1.3 antibody as a model, this cell culture system was shown to glycosylate the IgG Fc region in a similar manner to IgG isolated from serum. The neutral, core fucosylated biantennary oligosaccharides found are present in serum IgG and no novel carbohydrate sequences were detected. The degree of terminal agalactosylation was also similar to normal serum, in contrast to the increased levels found in rheumatoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(β1,2) Man(α1,6) Man(β1,4) antenna. Unambiguous identification of the exact glycosylation pattern of the antibody was carried out by a combination of specific exoglycosidase digestions, gel permeation chromatography of 2-aminobenzamide derivatives, high pH anion exchange chromatography and methylation analysis followed by gas–liquid chromatography-mass spectrometry. Abbreviations: CDR, complementarity determining region; CHO, chinese hamster ovary; GPC, gel permeation chromatography; 2-AB, 2-aminobenzamide; HPAEC-PAD, high pH anion exchange chromatography with pulsed amperometric detection; GC-MS, gas chromatography with mass spectrometry analysis; PNGase F, peptide-N-glycosidase F; PGC, porous graphitized carbon column; RAAM, reagent array analysis method; NeuAc: N-acetylneuraminic acid; NeuGc: N-glycolylneuraminic acid This revised version was published online in November 2006 with corrections to the Cover Date.     

Effects of PKCalpha activation on Ca2+ pump and KCa channel in deoxygenated sickle cells

We have previously shown that a pretreatment with phorbol12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC),reduced deoxygenation-induced K⁺loss and Ca²⁺ uptake and preventedcell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007,1995). The present study explores the detailed mechanism of thisPMA-induced inhibition. The main findings are, first, the detection ofPKC and PKC in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The -isoform only is translocated to the membrane and activated by PMAand by elevation of cytosolicCa²⁺. Second, PMA is demonstratedto activate Ca²⁺ efflux indeoxygenated SS cells by a direct stimulation of the Ca²⁺ pump. PMA, moreover, inhibitsdeoxygenation-induced, charybdotoxin-sensitive K⁺ efflux in SS cells. Thisinhibition is partly indirect and explained by the reduceddeoxygenation-induced rise in cytosolicCa²⁺ resulting fromCa²⁺ pump stimulation. However, asignificant inhibition of theCa²⁺-activatedK⁺ channels(K_Ca channels) by PMA can also bedemonstrated when the channels are activated byCa²⁺ plus ionophore, underconditions in which the Ca²⁺ pumpis operating near its maximal extrusion rate, but swamped byCa²⁺ plus ionophore. The data thussuggest a PKC-mediated phosphorylation both of theCa²⁺ pump and of theK_Ca channel or an auxiliaryprotein.

     

Temperature-Sensitive Initiation of Chromosome Replication in a Mutant of Bacillus subtilis

A mutant of Bacillus subtilis Ts37 has been isolated in which deoxyribonucleic acid (DNA) synthesis is inhibited at high temperature. The results presented here indicate that the process of initiation of DNA replication is temperature sensitive in this mutant. After shifting to 45 C, DNA increases 40 to 50% before synthesis ceases; an inhibition of protein synthesis permits an equivalent amount of DNA to be synthesized. A density shift experiment coupled with a marker frequency analysis shows that DNA synthesized at 45 C is highly enriched in the markers situated at the end of the chromosome. Transforming DNA extracted from a culture which has been incubated at 45 C exhibits the relative transforming efficiency for origin and terminus markers characteristic of completed chromosomes. After a shift back from 45 C to 30 C, reinitiation appears to occur always in the same region of the bacterial chromosome; in addition, replication as well as cell division is synchronized.     

Heat-induced changes in respiratory pathways and mitochondrial structure during microcycle conidiation of Neurospora crassa

Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46°C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25°C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding.     

Seasonal variations in size and morphology of Acanthocyclops robustus (Copepoda Cyclopoida)

The variations in the body length, swimming legs and some setaeof the freshwater cyclopid Acanthocyclops robustus were analysedduring an annual cycle in the field, and in laboratory experimentsat different culture temperatures. In Lake Créteil. ashallow temperate sand-pit lake, there is a seasonal morphologicalvariation of the setae: the plumose forms were restricted tothe warmer season and the spinose ones to colder periods. Contraryto other years, the specific ‘spine formula’ ofthe swimming legs (3-4-4-4) did not vary during the referenceyear. In laboratory experiments, both sex and post-embryonicdevelopmental temperatures had an effect on the length of adults.A significant parental effect on body length was detected. Pairswith anomalous spine formulae produced offspring either withanomalous spine formulae or typical 3-4-4-4 formulae. No significanteffects of parental culture temperatures, offspring culturetemperatures and sex on the total number of spines were found.Modification of plumose into spinose setae did not occur, whateverthe offspring developmental temperatures. The results presentedhere suggest that the alterations of the appendages of the cyclopidA.robustus could be compared to the cyclomorphosis in otherzooplanktonic groups, and contribute to our understanding ofthe role of phenotypic induction in aquatic biology.     

A new mutation, 3905insT, accounts for 4.8% of 1173 CF chromosomes in Switzerland and causes a severe phenotype

We have analysed 1173 cystic fibrosis (CF) chromosomes from Switzerland for eight mutations in the CF transmembrane conductance regulator (CFTR) gene. This permitted the identification of 88.5% of all mutations present. A novel insertion mutation in exon 20 of the CFTR gene, 3905insT, was discovered. This mutation accounted for 4.8% of CFTR gene mutations in Switzerland and has since been identified in other populations of probable Swiss descent. It is associated with a highly variable clinical phenotype but always with pancreatic insufficiency. Haplotype analysis with three intragenic microsatellites in the CFTR gene showed that the mutation is associated with a haplotype rarely identified on other CFTR alleles and, therefore, that the frequency of the mutation in Switzerland is explained by a founder effect of a relatively recent mutation event. Received: 17 February 1997 / Accepted: 26 March 1977     

The inhibitory effect of polymeric carboxylic amino-acids and urine on calcium oxalate crystallization

A new method has been established to follow the inhibitory effect of some polymeric-dicarboxylic-amino-acids and other poly-anionic derivatives. The technique is based on using calcium specific electrode to measure continuously free calcium ion activity in solution to assess the formation of calcium oxalate precipitate.The retardation effect of Poly-L-glutamic and aspartic acids has been established in 5–100 ppm range and compared to other monomeric and polymeric compounds with inactivated functional groups. The retardation effect is in agreement with previous reports on inhibition effect on crystal growth rate of seed crystals, with the same retardants.The inhibitory effect was also determined with normal urine and compared to pathological urine.