The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin

The relationship between synthesis and N-liked glycosylation site occupancy of recombinant human prolactin produced from C127 cells was studied with the aid of a battery of protein synthesis inhibitors. Non-lethal concentrations of sodium fluoride, gougerotin, puromycin, anisomycin, and emetine did not alter site occupancy, but low concentrations (<10g ml^–1) of cycloheximide increased the fraction of secreted prolactin bearing oligosaccharide from 20% to 80% of the total. Cycloheximide is an inhibitor of the elongation step of protein synthesis. The observed increase in glycosylation site occupancy upon addition of cycloheximide is consistent with the current opinion that the initial glycosylation event occurs cotranslationally during a limited time period. Cycloheximide may extend this time period by reducing elongation rate. However, the absence of any effect from treatment with other inhibitors of elongation suggests that cycloheximide is unique in its behavior on this system.Abbreviations clp-PRL clipped form of prolactin - DMEM/F12 11 Dulbecco's Modified Eagle's Medium/Ham's nutrient mixture F12 - G-PRL glycosylated (N-linked) fraction of prolaction - NG-PRL prolactin fraction without N-linked glycosylation - PMSF phenylmethylsulfonylfluoride     

On the inducibility of nitrate transport by tobacco cells

The question as to whether the nitrate transport system is induced by nitrate was addressed using a cell suspension of the XD line of Nicotiana tabacum L. cv. Xanthi as an experimental system. The cells were grown on area as the sole nitrogen source, and tungstate was used to render nitrate reductase non-functional. To avoid shock due to vacuum filtration, the cells, were harvested by gravity filtration. Nitrate uptake by cells, which were harvested, transferred to fresh medium, and immediately exposed to nitrate (freshly harvested cells), displayed a lag period of about 3 h.
In cells which were given incubation periods in fresh medium before exposure to nitrate (preincubated cells), the lag period was considerably shortened. After 3 h of preincubation in the absence of nitrate (recovered cells), the lag period was almost completely eliminated. Cycloheximide inhibited nitrate uptake by recovered cells within minutes, and prevented the development of nitrate uptake in freshly harvested cells. Cycloheximide did not affect uptake of α-aminoisobutyric acid (AIB) within the first 2 h after its addition. Recovery of the membrane potential from a low value just after the harvest of the cells to a maximal value 3 h later, was observed using the lipophilic cation methyltriphenylphosphonium (MTPP⁺), supplied at low concentrations, as a probe. Depolarization of the membrane potential by MTPP⁺, at the millimolar range, caused a rapid inhibition of nitrate uptake by recovered cells. The results indicate that nitrate transport by the XD cells depends on the membrane potential and on protein components with short half life. In addition, it requires a continuous protein synthesis. The effects of physical manipulation on nitrate uptake are discussed.     

Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

Functional binding of cardiolipin to cytochromec oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa ₃ complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K _d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [¹⁴C]-acetyl-cardiolipin (K _d <0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.     

Developmental regulation of an acyl carrier protein gene promoter in vegetative and reproductive tissues

The expression of an Arabidopsis acyl carrier protein (ACP) gene promoter has been examined in transgenic tobacco plants by linking it to the reporter gene -glucuronidase (GUS). Fluorometric analysis showed that the ACP gene promoter was most active in developing seeds. Expression was also high in roots, but significantly lower in young leaves and downregulated upon their maturation. Etiolated and light-grown seedlings showed the same level of GUS activity, indicating that this promoter is not tightly regulated by light. Histochemical studies revealed that expression was usually highest in apical/ meristematic zones of vegetative tissues. Young flowers (ca. 1 cm in length) showed GUS staining in nearly all cell types, however, cell-specific patterns emerged in more mature flowers. The ACP gene promoter was active in the stigma and transmitting tissue of the style, as well as in the tapetum of the anther, developing pollen, and ovules. The results provide evidence that this ACP gene is regulated in a complex manner and is responsive to the array of signals which accompany cell differentiation, and a demand for fatty acids and lipids, during organogenesis.     

Cell cycle mutation in Paramecium tetraurelia discriminates between sexual and vegetative functions

The temperature-sensitive mutation cc1 blocks a number of cell cycle processes in Paramecium including macronuclear DNA synthesis, oral morphogenesis, and the later stages of micronuclear mitosis. Oral morphogenesis and micronuclear mitosis also occur in the sexual pathway. This study shows that cc1 cells can proceed through conjugation or autogamy under restrictive conditions; neither stomatogenesis nor micronuclear mitosis is blocked. Fertilization and macronuclear determination occur normally, but DNA synthesis in macronuclear anlagen is blocked. Therefore, this mutation discriminates between oral replacement during meiosis and vegetative prefission stomatogenesis, and between mitotic spindle elongation during the pregamic and postzygotic divisions and spindle elongation during the vegetative cell cycle. These results point to a fundamental regulatory difference between morphogenesis in the vegetative and sexual pathways. © 1994 Wiley-Liss, Inc.     

Thermostable β-Glycosidase from Sulfolobus Solfataricus

The Sulfolobus solfataricus β-glycosidase (Sβgly) is a thermostable and thermophilic glycosyl-hydrolase with broad substrate specificity. The enzyme hydrolizes β-D-gluco-, fuco-, and galactosides, and a large number of /Winked glycoside dimers and oligomers, linked β1-3, β1-4, and β1-6, It is able to hydrolize oligosaccharides with up to 5 glucose residues. Furthermore, it is also able to promote transglycosylation reactions. The corresponding gene has been cloned and overexpressed both in yeast and Escherichia coli. Based on sequence and functional data, the Sβgly has been assigned to the so-called BGA family of glycosyl-hydrolases, including β-glycosidases, β-galactosidases and phosho-β-galactosidases from mesophilic and thermophilic organisms of the three domains. The Sβgly has been crystallized and the resolution of its structure is in progress. Because of its special properties, the enzymes has considerable biotechnological potential.     

The Different Behavior of Diphtheria Toxin, Modeccin and Ricin in HeLa Cells Infected with Trypanosoma cruzi

ABSTRACT. We have studied the action of diphtheria toxin, modeccin and ricin on HeLa cells infected by Trypanosoma cruzi . Parasitized HeLa cells were resistant to diphtheria toxin and modeccin, whereas non-parasitized cells from the same cultures and control cultures showed cytopathological alterations. Protein synthesis, assayed by the incorporation of labelled methionine, diminished in toxin-treated control cultures but remained unaltered in the infected ones, compared to synthesis by untreated infected cells. Ricin, on the other hand, is a toxin that enters the cytoplasm by endocytosis. It has greater cytopathological effects in parasitized cells than in non-parasitized ones from the same cultures or uninfected control cells. Protein synthesis was inhibited in infected cultures treated with ricin.