Sites of Tubulin Polymerization in PC 12 Cells

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.     

Control of Globodera tabacum solanacearum by Alternating Host Resistance and Nematicide

The feasibility of alternating use of resistant vs. susceptible flue-cured tobacco cultivars to improve control of Globodera tabacum subsp, solanacearum (TCN) was investigated at two Virginia locations in 1984-86. Post-harvest TCN population densities were reduced in each year of the study when fenamiphos was used with a TCN-resistant cultivar (NC 567), relative to susceptible cultivars (K 326 or Mc 944). Using NC 567 with fenamipbos also reduced preplant TCN population densities in the next growing season. Egg population densities before planting in 1986 were significantly lower in plots planted with NC 567 in 1984, even when a susceptible cultivar had been planted in 1985. Use of fenamiphos with NC 567 in 1984 and 1985 further reduced preplant egg population densities in 1986. Economic returns were significantly greater in 1984 when NC 567 was used with fenamiphos, rather than a susceptible cultivar. Treatments involving fenamiphos and (or) NC 567 in 1984 and 1985 resulted in higher economic returns in 1986 than did treatments using a susceptible cultivar without fenamiphos in both previous years. Economic returns were highest in 1986 when fenamiphos and NC 567 were used in 1984 and 1985 and a susceptible cultivar was planted in 1986.     

Altered biodistribution of indium-111-labeled monoclonal antibody 96.5 to tumors and normal tissues of nude mice bearing human melanoma xenografts in visceral organs

Summary Human melanoma xenografts were produced in the subcutis, kidney, cecum and liver of different nude mice. An¹¹¹In-labeled anti-(human melanoma) monoclonal antibody (96.5) or an¹¹¹In-labeled nonspecific control monoclonal antibody (ZCE-025) was injected intravenously in separate groups of mice. Radioactive antibody accumulation was measured in tumor, blood, viscera, and carcasses. mAb 96.5 targeted specifically to tumor tissue regardless of site of growth. Tumors in the liver exhibited significantly (P <0.05) higher tumor-to-blood ratios (45±6, mean ±SEM) than xenografts at other visceral organs, the lowest value being found for subcutaneous melanoma (2.6±0.5). The differences in tumor-to-blood ratio were due to significant alterations of antibody biodistribution, since the actual antibody concentration in the different tumor sites was similar. The percentage of recovered anti-melanoma antibody per milliliter of blood in mice with visceral lesions (4.6±1.1%/ml) was significantly lower than that found in mice with subcutaneous tumors (9.5±1.4%/ml,P <0.05). Moreover, significantly higher levels (18.2±3.2%/g, 31.0±5.1%/g, respectively) of the melanoma mAb 96.5 were found in normal liver and spleen tissue recovered from mice with visceral tumors as compared to tissue from mice with subcutaneous tumors (9.2±0.9%/g, 13.5±1.9%/g, respectively;P <0.05). These results demonstrate that the presence of visceral tumor can significantly affect tumor-to-blood ratios, blood levels, and biodistribution of¹¹¹In-labeled mAb 96.5.This work was supported in part by funds from the National Institutes of Health, National Cancer Institute, Grant R35-CA42107 and Core Grant CA16672     

The expression of a highly expressed Bacillus subtilis gene is not reduced by introduction of multiple codons normally not present in such genes

A recombinant DNA Proteus mirabilis L-form expression system, LVI (pJS127), was used to synthesize human fusion interferon alpha 1 (f-IFN-alpha 1). In the expression plasmid used, the complete coding sequence of IFN-alpha 1 was linked to the streptococcal speA promoter and the 5' end of the speA structural gene including its signal sequence coding region. LVI (pJS127) was capable of complete secretion into the culture medium of biologically active f-IFN-alpha 1 whose identity was confirmed by immunological and chemical evidence. In particular, bacterial L-forms were for the first time shown to be capable of correct signal peptide processing, as determined by N-terminal sequencing of the secreted f-IFN.     

The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution

Platelet factor 4 (PF4), which is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin. To date, six other proteins are known that are homologous in sequence with PF4 but have quite different functions. The structure of a tetramer of bovine PF4 complexed with one Ni(CN)4(2-) molecule has been determined at 3.0 A resolution and refined to an R factor of 0.28. The current model contains residues 24-85, no solvent, and one overall temperature factor. Residues 1-13, which carried an oligosaccharide chain, were removed with elastase to induce crystallization; residues 14-23 and presumably 86-88 are disordered in the electron density map. Because no heavy atom derivative was isomorphous with the native crystals, the complex of PF4 with one Ni(CN)4(2-) molecule was solved using a single, highly isomorphous Pt(CN)4(2-) derivative and the iterative, single isomorphous replacement method. The secondary structure of the PF4 subunit, from amino- to carboxyl-terminal end, consists of an extended loop, three strands of antiparallel beta-sheet arranged in a Greek key, and one alpha-helix. The tetramer contains two extended, six-stranded beta-sheets, each formed by two subunits, which are arranged back-to-back to form a "beta-bilayer" structure with two buried salt bridges sandwiched in the middle. The carboxyl-terminal alpha-helices, which contain lysine residues that are thought to be intimately involved in binding heparin, are arranged as antiparallel pairs on the surface of each extended beta-sheet.     

Characterization of Senescence in Lemna gibba G3: A Determinate Growth System

Frond senescence in Lemna gibba G3 was characterized, and itscontrol by light, ABA and kinetin investigated. The plant exhibitsa determinate growth pattern with a frond producing a set numberof daughter fronds before undergoing senescence and death regardlessof whether or not it flowers. When a frond was cut in half,the distal half (half frond) which lacks any meristem underwentrapid senescence as compared with intact fronds. In both intactand half fronds, the onset of senescence was accelerated byABA and retarded by kinetin. Continuous white light acceleratedsenescence in both intact and half fronds over the dark controls.Under different photoperiodic light regime, the pace of daughterfrond production is accelerated in proportion to the lengthof light period. In half fronds, however, very short photoperiodiclight treatments (e.g. 1L: 23D or 3L: 21D) rather delayed senescenceover the dark controls. Two separate light control systems operatingin opposite directions in Lemana senescence appear to exist. ¹Present address: Department of Biology, Yonsei University,Seoul 120-749, Korea ²Present address: U.S. Department of Agriculture, Aero SpaceBuilding, Rm. 323, 901 D Street, S.W., Washington, D.C. 20251-2200, U.S.A. (Received July 13, 1989; Accepted May 8, 1990)     

The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance

Mutagenized populations of Arabidopsis thaliana seedlings were screened for plants capable of root growth on inhibitory concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Four of the mutant lines recovered from this screen display a defect in root gravitropism as well as hormone resistance. The aerial portions of these plants are similar to wild-type in appearance. Genetic analysis of these four mutants demonstrated that hormone resistance segregated as a recessive trait and that all four mutations were alleles of the auxin-resistant mutation aux1 [Maher HP, Martindale SJB (1980) Biochem Genet 18: 1041-1053]. These new mutants have been designated aux1-7, 1-12, 1-15, and 1-19. The sensitivity of wild-type and aux1-7 roots to indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and ethylene was determined. The results of these assays show that aux1-7 plants require a 12-fold (indole-3-acetic acid) or 18-fold (2,4-dichlorophenoxyacetic acid) higher concentration of auxin than wild-type for a 50% inhibition of root growth. In addition, ethylene inhibition of root growth in aux1-7 plants is approximately 30% that of wild-type at saturating ethylene concentrations. These results indicate that aux1 plants are resistant to both auxin and ethylene. We have also determined the effect of ethylene treatment on chlorophyll loss and peroxidase activity in the leaves of aux1 and wild-type plants. No difference between mutant and wild-type plants was observed in these experiments, indicating that hormone resistance in aux1 plants may be limited to root growth. Our studies suggest that the AUX1 gene may have a specific function in the hormonal regulation of gravitropism.     

Bradykinin Stimulates Phosphoinositide Hydrolysis and Mobilization of Arachidonic Acid in Dorsal Root Ganglion Neurons

Cultures of fetal rat dorsal root ganglion neurons (7 days in culture) were prelabeled with myo-[3H]inositol or [3H]arachidonic acid for 24 h and stimulated with 10 microM bradykinin for time intervals of 5-300 s. The incubation was terminated by addition of 5% perchloric acid to extract inositol phosphates or organic solvent to extract lipids. Inositol phosphates were resolved by anion-exchange HPLC; lipids were resolved by TLC. Bradykinin stimulation resulted in a 10-fold increased accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol bisphosphate (IP2) (fivefold) by 5 s. The increase in IP3 was transient (half maximal by 1 min), whereas stimulated IP2 levels were sustained for several minutes. Even longer term increases were observed in inositol monophosphate. Stimulation also resulted in a threefold increase in arachidonic acid which was preceded by transient increases in diacylglycerol (twofold) and arachidonoyl-monoacylglycerol (threefold). The temporal lag in the accumulation of arachidonic acid with respect to diglyceride and monoglyceride suggested the involvement of di- and monoglyceride lipases in arachidonic acid mobilization. A role for phospholipase A2 is also possible, because pretreatment of cultures with quinacrine partially blocked arachidonic acid release. Bradykinin-stimulated arachidonic acid release was decreased in the presence of calcium channel blockers nifedipine or verapamil (50 microM), or EDTA (2.5 mM). The role of calcium was verified further in that accumulation of phosphatidic acid, diacylglycerol, and arachidonic acid was maximally stimulated by treatment with the calcium ionophore A23187 (20 microM).