Characterization of two maize HSP90 heat shock protein genes: expression during heat shock, embryogenesis, and pollen development.

We have isolated two genes from Zea mays encoding proteins of 82 and 81 kD that are highly homologous to the Drosophila 83-kD heat shock protein gene and have analyzed the structure and pattern of expression of these two genes during heat shock and development. Southern blot analysis and hybrid select translations indicate that the highly homologous hsp82 and hsp81 genes are members of a small multigene family composed of at least two and perhaps three or more gene family members. The deduced amino acid sequence of these proteins based on the nucleotide sequence of the coding regions shows 64-88% amino acid homology to other hsp90 family genes from human, yeast, Drosophila, and Arabidopsis. The promoter regions of both the hsp82 and hsp81 genes contain several heat shock elements (HSEs), which are putative binding sites for heat shock transcription factor (HSF) commonly found in the promoters of other heat shock genes. Gene-specific oligonucleotide probes were synthesized and used to examine the mRNA expression patterns of the hsp81 and hsp82 genes during heat shock, embryogenesis, and pollen development. The hsp81 gene is only mildly heat inducible in leaf tissue, but is strongly expressed in the absence of heat shock during the pre-meiotic and meiotic prophase stages of pollen development and in embryos, as well as in heat-shocked embryos and tassels. The hsp82 gene shows strong heat inducibility at heat-shock temperatures (37-42 degrees C) and in heat shocked embryos and tassels but is only weakly expressed in the absence of heat shock. Promoter-GUS reporter gene fusions made and analyzed by transient expression assays in Black Mexican Sweet (BMS) Maize protoplasts also indicate that the hsp82 and hsp81 are regulated differentially. The hsp82 promoter confers strong heat-inducible expression of the GUS reporter gene in heat-treated cells (60- to 80-fold over control levels), whereas the hsp81 promoter is only weakly heat inducible (5- to 10-fold over control levels).

[Interdisciplinary continuing education in palliative nursing, medicine and care]. / Interdisziplinäre Weiterbildung in Palliativpflege, -medizin und -betreuung.

Changing complementary goals in medicine, nursing and care pose new challenges to health professionals. The needs of the incurably ill or dying individuals and their relations are acknowledged more intensively again today. Quality of life and comprehensive care are corner-stones in the treatment and care of this group of patients. Realisation of the principles of palliative medicine and care demands a targeted approach to this multifaceted and burdening field. Nurses, doctors, social workers and other therapists have not yet been optimally prepared for this task. A common interdisciplinary training in this evolving field gives all the specialists from the different fields an opportunity for preparation to this task.

Intron-mediated enhancement of heterologous gene expression in maize.

Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5' translated region but not when it was located upstream of the promoter or within the 3' untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity and quality of mRNA.

Genetically transformed maize plants from protoplasts.

Genetically transformed maize plants were obtained from protoplasts treated with recombinant DNA. Protoplasts that were digested from embryogenic cell suspension cultures of maize inbred A188 were combined with plasmid DNA containing a gene coding for neomycin phosphotransferase (NPT II) next to the 35S promoter region of cauliflower mosaic virus. A high voltage electrical pulse was applied to the protoplasts, which were then grown on filters placed over feeder layers of maize suspension cells (Black Mexican Sweet) and selected for growth in the presence of kanamycin. Selected cell lines showed NPT II activity. Plants were regenerated from transformed cell lines and grown to maturity. Southern analysis of DNA extracted from callus and plants indicated the presence of the NPT II gene.

Factors affecting the level of kanamycin resistance in transformed sunflower cells.

A 230 base pair DNA segment containing the sequences 5' to the 700 to 750 nucleotide (nt) transcript 7' (ORF 3; RF Barker, KB Idler, DV Thompson, JD Kemp 1983 Plant Mol Biol 2: 335-350) of the octopine tumor inducing plasmid pTiA6 has been isolated. This region has (a) 180 base pairs of DNA upstream of the TATA box, (b) the start of RNA synthesis, and (c) the entire 5' untranslated region of the gene. We have fused this presumed promoter fragment to the neomycin phosphotransferase II (NPTII) gene from Tn5 in a plant expression cassette. After recombination into a tumor inducing plasmid delivery plasmid, this cassette confers selectable kanamycin resistance to transformed sunflower cells. Removal of the out-of-frame ATG in the 5' leader sequence of the NPTII gene by two different modifications increased both the levels of NPTII enzyme activity and the ID(50) for kanamycin in the tumor cells. The promoter region of the transcript 7 gene gives levels of kanamycin resistance equivalent to the nopaline synthase promoter and octopine synthase promoter when used in the same constructions and assayed in the same tissues.

Cryopreservation of Dictyocaulus viviparus third-stage larvae and Trichinella spiralis muscle larvae.

In cryopreservation studies with third-stage larvae of Dictyocaulus viviparus, best results were achieved by incubating larvae in 0.05% NaOCl at 37 degrees C to remove the sheath, followed by cooling at a rate of 1 degree C min per min down to about 0 degree C. After an equilibration time of 10 min at +4 degrees C with or without 4% polyethylene glycol-400 as cryoprotectant, samples were frozen at the same cooling rate to an intermediate temperature of -20 degrees C, maintained at this temperature for 10 min and finally plunged into liquid nitrogen for storage. Three groups of 3 calves were infected with the following batches of third-stage larvae: (a) fresh, sheated; (b) fresh, exsheathed; (c) exsheathed, cryopreserved for 13 weeks in liquid nitrogen and subsequently thawed. Although 62% of group (c) were regarded as viable in vitro, their infectivity to calves was low and only an average of 0.08% of the inoculated larvae (3000 per animal) developed into adult lungworms (= infectivity rate). Average infectivity rates of fresh, sheathed (a) and fresh, exsheathed (b) larvae were much higher (38.3% and 29.7%) and not significantly different from each other. Two of the calves inoculated with previously frozen larvae and all of the calves infected with fresh larvae excreted first-stage larvae in their faeces, but the latter groups in higher quantities. The results show that cryopreservation of exsheathed third-stage larvae of D. viviparus is possible, but for strain maintenance infection doses greater than 3000 larvae should be used for inoculation of calves.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prevention of nausea and emesis during cytostatic therapy. Antiemetic efficacy of high-dosage oral metoclopramide without and with prednisone]. / Nausea- und Emesis-Prophylaxe bei zytostatischer Therapie. Antiemetische Wirksamkeit von hochdosiertem oralem Metoclopramid ohne und mit Prednison.

In a randomized trial of 58 cancer patients receiving strongly emetogenic cytostatic drugs (cisplatin or comparable cytostatic agents, alone or in combination), the anti-emetic action of oral metoclopramide was tested, alone or combined with prednisone. Patients of group A (33 during 46 treatment cycles) received three times 50 mg metoclopramide and three times 25 mg prednisone. Those of group B (25 patients during 35 cycles) received three times 50 mg metoclopramide. The drug was given two hours before as well as two hours and six hours after administration of the cytostatic drugs. Good or satisfactory prophylaxis of nausea and vomiting (no or at most three vomits) was achieved during 37 cycles (80.5%) of group A patients and 30 (85.7%) of group B patients. Complete absence of vomiting was obtained in 52% of cycles in group A, 46% of cycles in group B; complete absence of nausea in 39% and 32%, respectively. There was no statistically significant difference between the two treatments in their preventive action. Three times 50 mg metoclopramide (with or without prednisone) four hours apart during one cytostatic-drug cycle was well tolerated by patients in both groups and can be recommended as a simple, practical prophylaxis of nausea and vomiting in the course of strongly emetogenic treatment of cancer.

Effective control of chemotherapy-induced nausea and vomiting with oral prednisone and metoclopramide.

Repeated oral doses of metoclopramide (50 mg) and prednisone (25 mg) completely prevented nausea and vomiting (N + V) in approximately 50% and substantially reduced N + V in an additional 27%-36% of 56 chemotherapy courses in 30 consecutive cancer patients who were receiving primarily cisplatin. Toxicity from this antiemetic regimen was minor. Simple oral N + V-prevention with metoclopramide and prednisone is as effective but less cumbersome and considerably less expensive than either high doses of intravenous corticosteroids and/or intravenous metoclopramide.

Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles.

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H(+)-transport as measured by [(14)C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP.Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn(2+) is almost as effective as Mg(2+), while Ca(2+) is ineffective. Excess divalent cations, particularly Ca(2+), reduces the pH gradient. H(+) transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with (36)Cl(-) indicate that ATP-driven H(+) transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, inhibit methylamine accumulation and (36)Cl(-) uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N'-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.

Localization of the proton pump of corn coleoptile microsomal membranes by density gradient centrifugation.

Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [(14)C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [(14)C]SCN(-) uptake and (36)Cl(-) uptake activities coincided with [(14)C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin-stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker.