Asn-linked oligosaccharides in lectin-resistant tumor-cell mutants with varying metastatic potential

MDW4, a wheat germ agglutinin-resistant mutant of the metastatic murine tumor MDAY D2 has previously been shown to be poorly metastatic when injected intravenously and non-metastatic when injected subcutaneously into syngeneic mice. W4EB8, a Bandeiraea simplicifolia (BSII) lectin-selected subline of MDW4 has previously been shown to be intermediate between that of MDAY-D2 and MDW4 cell for sensitivity to lectin and metastatic phenotype when injected intravenously into mice. The Asn-linked oligosaccharides from MDAY-D2, MDW4 and W4EB8 cells were released enzymatically with peptide N-glycosidase, reduced with tritiated sodium borohydride and fractionated by Concanavalin-A--Sepharose affinity chromatography and high-performance liquid chromatography (HPLC). Structures of the major fractions were determined by a combination of glycosidase digestion and sizing, gas-liquid chromatography/mass spectrometry and by proton nuclear magnetic resonance. Wild-type and mutant cells processed high-mannose-type structures to biantennary (GlcNAc)2(Man)3(GlcNAc)2. In MDAY-D2 cells this structure was processed further to sialylated tetra-antennary complex with polylactosamine-containing antennae terminating in either sialic acid or alpha 1-3-linked galactose. MDW4 cells had four or five times more (GlcNAc)2(Man)3(GlcNAc)2 than MDAY-D2 cells and a major component of tri-antennary (GlcNAc)3(Man)3(GlcNAc)2 (i.e. 2,2,6-substituted tri-mannosyl core) that was not found in wild-type cells. The partial revertant, W4EB8 had intermediate levels of mutant (GlcNAc)3(Man)3(GlcNAc)2 and sialylated complex-type carbohydrates. The results indicate that a shift in expression from incomplete complex type to sialylated tri/tetra-antennary complex-type carbohydrates in tumor cell may enhance the metastatic potential of tumor cells in the experimental metastasis assay. In addition, somatic cell hybridization analysis indicated that the defect in MDW4 cells was identical to that of the Chinese hamster ovary mutant Lec8: a deficiency in UDP-galactose transport into the golgi.     

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes

Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 microgram/ml insulin, 3 X 10(-7) M linoleic acid, 1 X 10(-8) M H2SeO3) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham's F12, 1% FBS + deoxycytidine + BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.     

Biosynthesis of lipophosphoglycan from Leishmania donovani: characterization of mannosylphosphate transfer in vitro.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes and is composed of a capped polymer of repeating PO4-6Gal(beta 1,4)Man alpha 1 disaccharide units linked via a phosphosaccharide core to a lyso-1-O-alkylphosphatidylinositol anchor. An exogenous acceptor composed of the glycolipid anchor portion of LPG was shown to stimulate the enzymatic synthesis of the repeating phosphorylated disaccharide units of LPG in a cell-free system. Using the exogenous acceptor, GDP-[3H]Man, [beta-32P]GDP-Man, and unlabeled UDP-Gal as substrates, membrane preparations from an LPG-defective mutant of L. donovani that lacks endogenous acceptors catalyzed the incorporation of the doubly labeled mannosylphosphate unit into a product that exhibited the chemical and chromatographic characteristics of LPG. Analysis of fragments generated by mild acid hydrolysis of the radiolabeled product indicated that [3H]mannose-1-[32P]PO4 had been transferred from the dual-labeled sugar nucleotide. These results are consistent with the proposal that the repeating units of the L. donovani LPG are synthesized by the alternating transfer of mannose 1-phosphate and galactose from their respective nucleotide donors.     

Mutations in the DNA gyrB gene that are temperature sensitive for lambda site-specific recombination, Mu growth, and plasmid maintenance.

We report the isolation of two mutations in the gyrB gene of Escherichia coli K12 obtained from an initial selection for resistance to coumermycin A1 and a subsequent screening for bacteria that fail to support site-specific recombination of phage lambda, i.e., Him-. These two mutations have a temperature-sensitive Him- phenotype, supporting site-specific recombination efficiently at low temperature, but inefficiently at high temperatures. Like other Him mutants, the gyrB-him mutants fail to plate phage Mu; again this defect is observed only at high temperatures. Additional thermally sensitive characteristics have also been observed; growth of lambda as well as maintenance of the plasmids pBR322 and F' gal are reduced at high temperature. Restriction of foreign DNA imposed by a P1 prophage is also reduced in these mutants. The temperature-sensitive phenotypic characteristics imposed by both the gyrB-him-230(Ts) and gyrB-him-231(Ts) mutations correlate with in vitro studies that show decreased gyrase activity, especially at higher temperatures, and in vivo studies showing reduced supercoiling of lambda DNA in the mutants at high temperature.     

Amino acid sequence differences in the alpha chains of pea seed isolectins: C-terminal processing

The complete amino acid sequence of the alpha chains of both isolectins found in pea seeds has been determined using automated Edman degradation. We show that the alpha chains of these two proteins differ only at their C-termini: isolectin B is two amino acids longer than isolectin A. Furthermore, the alpha chains of both isolectins are shorter than would be predicted from the nucleotide sequence of a cDNA clone for pea lectin. We suggest, therefore, that these proteins arise from differential C-terminal processing. Amino acid composition data and C-terminal analysis show that the beta chains have also been processed at their C-termini, but in this case identical chains for both isolectins are produced.     

A Prototype Recombinant-Protein Based Chlamydia pecorum Vaccine Results in Reduced Chlamydial Burden and Less Clinical Disease in Free-Ranging Koalas (Phascolarctos cinereus)

Diseases associated with Chlamydia pecorum infection are a major cause of decline in koala populations in Australia. While koalas in care can generally be treated, a vaccine is considered the only option to effectively reduce the threat of infection and disease at the population level. In the current study, we vaccinated 30 free-ranging koalas with a prototype Chlamydia pecorum vaccine consisting of a recombinant chlamydial MOMP adjuvanted with an immune stimulating complex. An additional cohort of 30 animals did not receive any vaccine and acted as comparison controls. Animals accepted into this study were either uninfected (Chlamydia PCR negative) at time of initial vaccination, or infected (C. pecorum positive) at either urogenital (UGT) and/or ocular sites (Oc), but with no clinical signs of chlamydial disease. All koalas were vaccinated / sampled and then re-released into their natural habitat before re-capturing and re-sampling at 6 and 12 months. All vaccinated koalas produced a strong immune response to the vaccine, as indicated by high titres of specific plasma antibodies. The incidence of new infections in vaccinated koalas over the 12-month period post-vaccination was slightly less than koalas in the control group, however, this was not statistically significant. Importantly though, the vaccine was able to significantly reduce the infectious load in animals that were Chlamydia positive at the time of vaccination. This effect was evident at both the Oc and UGT sites and was stronger at 6 months than at 12 months post-vaccination. Finally, the vaccine was also able to reduce the number of animals that progressed to disease during the 12-month period. While the sample sizes were small (statistically speaking), results were nonetheless striking. This study highlights the potential for successful development of a Chlamydia vaccine for koalas in a wild setting.     

The terminal respiratory chain of the methylotrophic bacterium Methylophilus methylotrophus

Cytochrome oxidase o has been isolated from the obligately aerobic, methylotrophic bacterium Methylophilus methylotrophus in the form of a cytochrome cL-o complex. The latter is comprised of cytochrome cL (Mr 21 000) and cytochrome o (Mr 29 000) in a 1-2:1 ratio, possibly in association with one or more minor polypeptides; the complex exhibits a high ascorbate-TMPD oxidase activity which is inhibited non-competitively by cyanide (Ki approximately 2 microM). In contrast, the oxidation of methanol by whole cells is inhibited uncompetitively by cyanide (Ki approximately 4 microM), thus indicating the involvement in methanol oxidation of cytochrome oxidase aa3 rather than o.