The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Helminths of rodents and marsupials from Papua New Guinea,with the description of two new species,Echinostoma echymiperae n. sp. (Digenea: Echinostomatidae) and Vampirolepis peroryctis n. sp. (Cestoda: Hymenolepididae)

The following rodents and marsupials from the Western Highlands of Papua New Guinea have been examined for helminths: Anisomys imitator, Melomys spp., Pogonomelomys ruemmleri, Rattus spp., Echymipera kalubu and Peroryctes raffrayanus. Two new species and a number of new host records are reported. Echinostoma echymiperae n. sp., a digenean from the intestine of Echymipera kalubu, is characterised by the number of collar spines, the body armature and the shape and position of the gonads. Vampirolepis peroryctis n. sp., a cestode from the intestine of Peroryctes raffrayanus, is characterised by the length of the rostellar hooks, the shape of the ovary, the arrangement of the testes in a triangle and the extent of the cirrus-sac. Hymenolepis aklei, H. bradleyi, H. antechini, H. bettongiae, H. cercarteti, H. isoodontis and H. potoroi are transferred to Vampirolepis as new combinations. E. kalubu is a new host for Linstowia semoni and Pogonomelomys ruemmleri is a new host for Hymenolepis diminuta. V. peroryctis is the first platyhelminth to be reported from Peroryctes raffrayanus and Raillietina (Raillietina) sp. the second to be reported from the genus Melomys.     

Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens.

Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E'o = -233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c3 group, which is supported by N-terminal sequence data up to the first heme binding site.     

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.     

Mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress

An in vitro flow apparatus in combination with cultured endothelium was used to determine the effects of fluid-generated shear stress on cells undergoing mitosis and cytokinesis. Cell responses were recorded by time-lapse video microscopy under phase contrast or Hoffman modulation contrast optics. Completion of cell division in mitotic cells was dependent upon both the initial presence of intercellular attachments and the magnitude of fluid wall shear stress. In nonisolated populations, 95.3%, 69.5%, and 57.1% of the cells completed cell division as opposed to 66.6%, 20.4%, and 11.9% in the isolated cell groups at 2.8, 14.1, and 33 dynes/cm², respectively. Prestressing cells for 14 h prior to monitoring failed to increase retention of isolated mitotic cells. The presence of neighboring cells facilitated replication by providing an anchoring attachment or a luminal surface for completion of division. Cell detachment most commonly occurred at the onset of cytokinesis when substrate contact areas were minimal and focal contacts were absent. A comparison between no flow controls and shear stress specimens indicated no significant differences in transit times for mitosis and cytokinesis. Thus, subconfluent endothelial cells may be more susceptible to detachment during cell division due to increases in shear stress, the absence of intercellular attachments, and reduced cell-substrate contacts. © 1994 wiley-Liss, Inc.     

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5–15 cm H₂O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: (1) Cells exposed to control (3 mm H₂O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H₂O for 7 days. (2) Conditioned medium from endothelial cells exposed to 10 cm H₂O for 7 days contained at ansferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. (3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure. © 1993 Wiley-Liss, Inc.     

Differences in Body Image and Depression Among Obese Women With and Without Binge Eating Disorder

Obese individuals with binge eating disorder (BED) differ from obese non-binge eating (NBE) individuals in a number of clinically relevant ways. This study examined attitudinal responses to various measures of body image in women seeking obesity treatment, by comparing NBE participants (n=80) to those with BED (n=48). It was hypothesized that women with BED would demonstrate greater attitudinal disturbance of body image compared to NBE individuals. It was further hypothesized that significant differences between groups would remain after statistically controlling for degree of depression. Consistent with the primary hypothesis, BED participants reported significantly increased attitudinal disturbance in body dissatisfaction and size perception compared to NBE participants. Although shared variance was observed between measures of depression and body image on some items, several aspects of increased body image disturbance remained after statistically controlling for depression. Treatment implications and recommendations for future research are discussed.     

Tripeptides as selective inhibitors of src-SH2 phosphoprotein interactions

Summary This paper describes the synthesis of phosphorylated peptides of the general structural Ac-Tyr(PO₃H₂)-Glu-Xaa_NH₂, where Xaa represents a hydrophobic -amino acid of d-configuration. These peptides displayed activities in the micromolar range in inhibiting src-SH2 domain/epidermal growth factor receptor interactions.     

Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td⁺, whereas particles produced after infection with td⁻ had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from td^ts to td⁺ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both td^ts and frd^ts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frd^ts mutants to frd⁺ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.     

Duration of Hydrogen Formation by Anabaena cylindrica B629 in Atmospheres of Argon, Air, and Nitrogen

The time course of hydrogen formation by Anabaena cylindrica was followed beneath an argon atmosphere alone and also beneath atmospheres of argon, nitrogen, and air in the presence of carbon monoxide (0.2%) and acetylene (5%). Hydrogen production beneath argon alone was comparable in rate and duration (7 to 12 days) to that which occurred beneath air in the presence of carbon monoxide (0.2%) and acetylene (5%). However, much greater longevity (16 to 26 days) and improved rates of hydrogen formation were obtained when algae were incubated beneath argon and particularly nitrogen, each supplemented with carbon monoxide and acetylene. The total hydrogen produced by these cultures was up to three times as much as that released by cultures incubated beneath argon alone. Hydrogen-oxygen ratios for argon cultures either with or without carbon monoxide and acetylene were initially 1:5 but approximated 1:2 when measured over the entire incubation period. In each case oxygen production and nitrogenase activity (acetylene reduction) continued at reduced rates after hydrogen evolution had ceased. The effects of methionine sulfoximine (2 μM), ammonium ions (0.5 mM), or both on oxygen production were generally negligible, while effects on hydrogen production were variable depending on the atmosphere used; in most cases, eventual destabilization of the system occurred. A brief comparison was made of the time courses of anaerobic and aerobic hydrogen formation by the marine cyanobacterium Calothrix membranacea. It was found that shaking of cultures was beneficial for hydrogen production but not strictly necessary. It is concluded that hydrogen production by A. cylindrica in air and particularly nitrogen in the presence of carbon monoxide and acetylene offers the best potential of the atmospheres considered on the basis of four criteria: rates and longevity of hydrogen formation, practicality of the atmosphere used, and tolerance of hydrogen evolution to slight changes in composition of the atmosphere.