Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors.

A single metabolic path leading to synthesis of ether lipids is known in animal cells, the major products of which are plasmalogens. To learn whether this peroxisomal path is also responsible for the synthesis of base-resistant lipid components of glycosylphosphoinositol (GPI)-anchored membrane proteins, we have investigated the structure of anchor precursor mannolipids both in wild-type cells (CHO-K1 and a macrophage-like line, RAW 264.7) and in two corresponding mutant cells in which ether lipid biosynthesis is severely impaired. We observe that the precursor mannolipids of both the wild-type and mutant cells do not include alkylglycerol. Nevertheless, both wild-type and mutant cells express cell surface GPI-anchored placental alkaline phosphatase (AP) which includes alkali-resistant hydrophobic chains in its anchor moiety. Thus, (i) in normal AP GPI anchor synthesis, any ether-linked substituents must be added either immediately before, during, or after anchor addition to AP, and (ii) the classical peroxisomal path for ether lipid synthesis appears not to contribute to the synthesis of GPI anchors.     

Purification of lectins from the stems of peanut plants

The stem of the peanut plant contains two lectins, a methyl -mannoside specific lectin (SL-I) and a lactose/cellobiose specific lectin (SL-II). These lectins are found to be developmentally regulated and maximum activites are observed in 3–4-weeks-old plants. The two lectins SL-I and SL-II have been purified from 3-week-old stem by affinity chromatography on Sephadex G-50 and guar gum matrices respectively. Both are glycosylated lectins and have the identical subunit molecular weight of 31 kDa.     

Hormonotoxins: the role of positive charge of lysine residue on the immunological,biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates

Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occured through the -NH₂ groups of -subunit of oLH as judged from RP-HPLC analysis. A 11 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.Abbreviations BSA Bovine Serum Albumin - CMC Carboxy methyl Cellulose - DTT Dithiothreitol - DMEM Dulbeco's Modified Eagle's Medium - DTNB Ellman's reagent [5,5-dithio-bis-(2-nitrobenzoic acid)] - EDTA Ethylenediaminetetraacetic acid - FPLC Fast Protein Liquid Chromatography - FCA Freund's Complete Adjuvant - FCS Fetal Calf Serum - Gelonin-30 Gelonin modified by SPDP - GnRH Gonadotropin-Releasing Hormone - Gelonin-SPDP SPDP modified derivative of gelonin - HEPES (N-[2-hydroxyethyl] piperazine-N-[-2-ethanesulphonic acid]) - IFA Incomplete Freund's Adjuvant - 2IT 2-Iminothiolane - IODOGEN 1,3,4,6-tetrachloro 3,6-diphenylglycouril - oLH Ovine Luteinizing Hormone - oLH-SPDP SPDP modified derivative of oLH - oLH-10 oLH modified by 2IT - oLH2IT Molar ratio of oLH and 2IT - PDP 2-Pyridyl-dithiopropionate - PAP Pokeweed Antiviral Protein - RIP Ribosome Inactivating Protein - RP-HPLC Reverse-Phase High Performance Liquid Chromatography - RPMI Roswell Park Memorial Institute - RIA Radioimmunoassay - RRA Radioreceptor Assay - SPDP N-Succinimidyl-3(2-pyridyldithio)propionate - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - TCA Trichloroacetic acid - TFA Trifluroacetic acid     

Isolation and characterization of temperature-sensitive mutants ofAnabaena variabilis impaired in nitrogen fixation

Temperature-sensitive (ts) mutants of the cyanobacteriumAnabaena variabilis ATCC 29413 were isolated following mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and post-treatment with metronidazole at 40°C. Of the 8000 clones isolated and tested, six mutants were conditionally lethal at the restrictive temperature (40°C). All the ts mutants exhibited differences in their rates of growth, chlorophyll content, pigment (phycocyanin and/or chlorophyll) ratios, heterocyst frequency, oxygen evolution and nitrogenase activity at the permissive temperature (28°C). A gradual loss of all the above features occurred after a period of 3 d at 40°C, followed by lysis of the cultures. Cessation of nitrogenase activity was found to be different in the different ts mutants. The temperature-sensitive nature of the mutants is suggested to be due to an impairment in iron metabolism since addition of ferric citrate to cultures at 40°C restored the ability to grow, produce heterocysts and fix nitrogen.     

Localization of the two major allergens in rye-grass pollen using specific monoclonal antibodies and quantitative analysis of immunogold labelling

Summary The intracellular localization of the two major allergens, Lol p I and Lol p IX, in rye-grass anthers was examined using monoclonal antibodies FMCA1 (specific for Lol p I) and FMCA7 (specific for Lol p IX) with immunocytochemical techniques and quantitative analysis. A newly developed anhydrous fixation technique in a mixture of glutaraldehyde, paraformaldehyde and 2, 2-dimethoxypropane followed by embedding in LR Gold resin resulted in both improved infiltration of pollen grains compared with existing techniques and the localization of these water-soluble antigens in their original sites compared with diffusion artefacts following aqueous methods. After anhydrous fixation, Lol p I was predominantly located in the electron-opaque regions of the cytosol of the vegetative cell of the tricellular pollen grains (24 counts m^-2), whereas Lol p IX was detected mainly within starch granules (16 counts m^-2). For both Lol p I and Lol p IX, similar labelling was detected in the cells of the endothecium and middle layer (18 counts m^-2), but none was found in the tapetal cells or orbicules.     

Sublethal malathion induced changes in the ovary of an air-breathing fish,Heteropneustes fossilis: a histological study

This paper describes effects of a sublethal (1.2 mg 1^–1) organophosphate, malathion, on the ovary of an air breathing catfish, Heteropneustes fossilis. The study focuses on microscopic changes that occur on ovigerous lamellae, oocytes at different stages of development and the nucleus of the immature oocyte. Also, change in estrogen levels in blood serum is investigated. Clumping of cytoplasm appears after 24 h of exposure to malathion. Clumping intensified after 48 h. Degeneration in the follicular cells was also observed. After 72 h exposure the number of nucleoli increased, nuclear materials shrunk, oocytes became adhered. With 96 h of exposure, nuclear materials of all the oocytes shrunk to a smaller clump. The oocytes fused together, and follicular epithelium became loose and ruptured. A few atretic oocytes were visible. Radioimmunoassay of the estrogen level in blood serum after 72 h of exposure of malathion showed a reduction in the level. This study showed that the histopathological condition of the gonad is reflected in malfunctioning of the endocrine system and hormonal disbalance.     

Banded krait minor satellite (Bkm) contains sex and species-specific repetitive DNA

A novel class of repetitive DNA was isolated from a Bkm DNA library by exclusion hybridization. This sequence was mapped to the short arm of the W chromosome of banded krait, Bungarus fasciatus. Southern blot hybridization showed that these sequences are sex and species specific. Sequence analysis of a 206 bp long clone, BR87, revealed the presence of a tandem array of two internal repeat units of 18–19 bp alternating with each other with a gap of 1,2 or 3 nucleotides. To our knowledge, this is the first report of an exclusively W chromosome-and species-specific repeat isolated from any reptile. The functional significance of this sequence based on its organisation is discussed.     

Scaleup of spinfilter perfusion bioreactor for mammalian cell retention

A spinning cylindrical filter, known as a spinfilter, permits the mammalian cell bioreactor operation at high perfusion rates leading to very high cell densities (10(7) mL(-1)). Filter screens with openings (25 mum) slightly larger than the average cell size have been used to retain single cells in suspension over a long period of operation without clogging. We have previously shown why it is necessary to optimize the rotational speed of the spinfilter in order to achieve efficient cell retention and avoid potential screen clogging. Effects of bulk mixing and perfusion rate on screen fouling and cell retention were also investigated. Based on this analysis, in this article, we suggest strategies for scaleup of spinfilters. Experimental data from 12- and 175-L (working volume) bioreactors is shown in support of the scaleup analysis. (c) 1994 John Wiley & Sons, Inc.     

Temperature Effects on Development and Reproduction of Heterodera cajani on Pigeonpea

Effects of constant and fluctuating temperature on development and reproduction of Heterodera cajani were studied on pigeonpea cv. ICPL 87 in growth chambers at 10, 15, 20, 25, and 30 C and in a greenhouse fluctuating between 22.2 and 37.8 C. Nematode penetration was greatest (P = 0.001) in roots at 25 C; there was no penetration at 10 C. The basal threshold temperature for development was calculated to be 11 C. Completion of one H. cajani generation required 17, 28, 35, and 66 days (323, 392, 315, and 264 degree-days) at 30, 25, 20, and 15 C, respectively, and 19 days (356 degree-days) at a fluctuating temperature. Survival was greater at 20 and 25 C than at 15 and 30 C. The greatest (P = 0.05) number of females (17.9 females per root) were produced at 25 C, compared with 13.2 at 20 C, 7.9 at 30 C, and 2.5 females at 15 C. Nematode reproduction was 1.6 to 7.1 times greater at 25 C than at other temperatures. Emergence of juveniles from egg sacs and cysts was greater at 25 and 30 C than at 15 and 20 C. Equations were developed to predict nematode development rate, cumulative juvenile emergence from egg sacs and cysts, and population increases as influenced by temperature.     

Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.