Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc₃Man₉(GlcNAc)₂, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man_6-9[GlcNAc]₂), mannosidase II (removes mannose residues from GlcNAcMan₅[GlcNAc]₂), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.     

Growth of the extreme thermophile Sulfolobus acidocaldarius in a hyperbaric helium bioreactor

The relationship between pressure and temperature as it affects microbial growth and metabolism has been examined only for a limited number of bacterial species. Because many newly-discovered, extremely thermophilic bacteria have been isolated from pressurized environments, this relationship merits closer scrutiny. In this study, the extremely thermophilic bacterium, Sulfolobus acidocaldarius, was cultured successfully in a hyperbaric chamber containing helium and air enriched with 5% carbon dioxide. Over a pressure range of approximately 1-120 bar and a temperature range of 67-80 degrees C, growth was achieved in a heterotrophic medium with the air mixture at partial pressures up to 3.5 bar. Helium was used to obtain the final, desired incubation pressure. No significant growth was noted above 80 degrees C over the same range of hyperbaric pressures, or at 70 degrees C when pressure was applied hydrostatically. Growth experiments conducted under hyperbaric conditions may provide a means to study these bacteria under simulated in situ conditions and simultaneously avoid the complications associated with hydrostatic experiments. Results indicate that hyperbaric helium bioreactors will be important in the study of extremely thermophilic bacteria that are isolated from pressurized environments.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Purification and properties of arylmannosidases from mung bean seedlings and soybean cells

Two arylmannosidases (signified as A and B) were purified tohomogeneity from soluble and microsomal fractions of mung beanseedlings. Arylmannosidase A from the microsomes appeared thesame on native gels and on SDS gels as soluble arylmannosidaseA, the same was true for arylmannosidase B. Sedimentation velocitystudies indicated that both enzymes were homogeneous, and thatarylmannosidase A had a molecular mass of 237 kd while B hada molecular mass of 243 kd. Arylmannosidase A showed two majorprotein bands on SDS gels with molecular masses of 60 and 55kd, and minor bands of 79, 39 and 35 kd. All of these bandswere N-linked since they were susceptible to digestion by endo-glucosaminidaseH. In addition, at least the major bands could be detected byWestern blots with antibody raised against the xylose moietyof N-linked plant oligosaccharides, and they could also be labeledin soybean suspension cells with [2–3H]mannose. ArylmannosidaseB showed three major bands with molecular masses of 72, 55 and45 kd, and minor bands of 42 and 39 kd. With the possible exceptionof the 45 and 42 kd bands, all of these bands are glycoproteins.Arylmannosidases A and B showed somewhat different kineticsin terms of mannose release from high-mannose oligosaccharides,but they were equally susceptible to inhibition by swainsonineand mannostatin A. Polyclonal antibody raised against the arylmannosidaseB cross-reacted equally well with arylmannosidase A from mungbean seedlings and with arylmannosidase from soybean cells.However, monoclonal antibody against mung bean arylmannosidaseA was much less effective against arylmannosidase B. Antibodywas used to examine the biosynthesis and structure of the carbohydratechains of arylmannosidase in soybean cells grown in [2–³H]mannose.Treatment of the purified enzyme with Endo H released 50% ofthe radioactivity, and these labeled oligosaccharides were ofthe high-mannose type, i.e. mostly Man9GlcNAc. The precipitatedprotein isolated from the Endo H treatment still contained 50%of the radioactivity, and this was present in modified structuresthat probably contain xylose residues. Mung beans mannosidases glycoproteins -soybean--mannosidases xylose-containing N-linked glycoproteins     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.     

Triggering of cytotoxic T lymphocytes and NK cells via the Tp103 pathway is dependent on the expression of the T cell receptor/CD3 complex

The expression and function of the T cell activation molecule Tp103 on human cloned cytotoxic CD3+ and CD3- cells were studied. All in vitro growing CD3+ and CD3- clones expressed Tp103 regardless of their phenotype and the expression of a CD3-associated TCR complex. Whereas the CD2 pathway was functional in all these clones, only CD3-expressing clones could be triggered via Tp103 to kill target cells. In contrast, both CD2 and Tp103 pathways were suppressed after modulation of the TCR complex with anti-CD3 mAb. This indicates that the function of Tp103 but not of CD2 is dependent on the expression of a functional Ag receptor on cytotoxic T cells. Furthermore, modulation of the Ag receptor induces a state of unresponsiveness in cytotoxic T cells that cannot be attributed to just the removal of the CD3/TCR complex from the cell membrane.     

Effects of progesterone on gonadotropin-releasing hormone receptor concentration in cultured estrogen-primed female rat pituitary cells

Acute (0.5–4 h) treatment of estradiol (E)-primed female rat pituitary cells with progesterone (P) augments gonadotropin-releasing hormone (GnRH)-induced LH release, whereas chronic (48 h) P-treatment reduces pituitary responsiveness to the hypothalamic decapeptide. Dispersed E-primed (48 h, 1 nM) rat pituitary cells were cultured for 4 or 48 h in the presence of 100 nM P to assess the effects of the progestagen on GnRH receptors and on gonadotrope responsiveness to the decapeptide. P-treatment (4 h) significantly augmented GnRH-receptor concentrations (4.44 ± 0.6 fmol/10⁶ cells) as compared to cells treated only with E (2.6 ± 0.5fmol/10⁶ cells). Parallel significant changes in GnRH-induced LH secretion were observed. The acute increase in GnRH-receptor number was nearly maximal (180% of receptor number in cells treated with E alone) within 30 min of P addition. Chronic P-treatment (48 h) significantly reduced pituitary responsiveness to GnRH as compared to E-treatment. The GnRH-receptor concentrations (3.9 ± 0.6 fmol/10⁶ cells), however, remained elevated above those in E-primed cells. GnRH-receptor affinity was not influenced by any of the different treatments. These results indicate that the acute facilitatory P-effect on GnRH-induced LH release is at least chronologically closely related to an increase in GnRH-receptor concentration. The chronic negative P-effect on pituitary responsiveness to GnRH, however, shows no relation to changes in available GnRH receptors.