35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

In vivo pH measurement in the xylem of broad-leaved trees using ion-sensitive field-effect transistors

Summary A new method of in vivo pH determination in the xylem of broad-leaved trees using ion-sensitive field effect transistors is developed and its suitability for use is studied. In the first few hours after the sensor had been implanted in the xylem signals could be detected which were generated in response to mechanical damage; particularly strong signal changes are detectable in Populus balsamifera L., Tilia cordata Mill, and Aesculus hippocastanum L. The pH values of the xylem sap extracted from branches corresponded to the values measured by the in vivo method only at certain times. Due to sensor drift the measuring accuracy of long-term experiments lasting up to 3 weeks is restricted. The in vivo measurement of pH in the xylem of poplar branches revealed the ability of the living xylem to buffer the pH of the sap to its own characteristic value.Dedicated to Prof. Dr. O. L. Lange to his 65th birthday     

Characterization of microcarrier cultures of neurons and astrocytes from cerebral cortex and cerebellum

In the present investigation a method is described for culturing cerebellar granule cells (glutamatergic neurons), cerebral cortical neurons (GABAergic neurons) and cortical astrocytes on Cytodex 3 microcarriers. It was possible to obtain a high yield of attached neurons and astrocytes on the microcarriers and the cell specific characteristics such as the ability to release neurotransmitter (neurons) and a high activity of glutamine synthetase (astrocytes) were preserved. This system, allowing mixtures of neurons and astrocytes at any given ratio to be produced, may constitute an attractive model system by which the interaction between neurons and astrocytes with regard to exchange of neurotransmitter precursors as well as other compounds may be studied.     

Asymmetric distribution of phospholipids in spectrin-poor erythrocyte vesicles

We have investigated by electron spin resonance, at 37 degrees C, the outside-inside passage and the equilibrium distribution of spin-labeled phospholipids, respectively, in ATP-containing ghosts, in heat-treated erythrocytes, and in heat-induced vesicles. The heat-treated vesicles were spectrin depleted to approximately 25% of the original content and had lost almost 100% of the other cytoskeletal proteins. Yet the vesicles, as long as they contained ATP, were capable of translocating the aminophospholipids with the same efficiency as the heat-treated erythrocytes, and almost with the same efficiency as ATP-containing ghosts. In the vesicles, sphingomyelin and phosphatidylcholine analogues underwent a very slow transverse diffusion as in native cells. We conclude that spectrin and other cytoskeleton proteins are not major factors for the establishment and maintenance of phospholipid asymmetry in human erythrocytes, which may be chiefly due to the aminophospholipid translocase activity.     

The role of CD18 in phorbol ester-induced human monocyte-mediated cytotoxicity

Monocyte cell surface molecules play an important role in the regulation of monocyte function. To investigate the molecular basis of monocyte-mediated cytotoxicity, we tested the ability of a variety of mediators to stimulate human monocyte-mediated cytotoxicity. Phorbol myristic acetate (PMA) stimulated significant monocyte-mediated killing of tumor cells in an 18-hr indium-111 release assay. Five other cytoactive substances did not induce monocyte-mediated cytotoxicity. The acquisition of monocyte cytotoxicity was associated with nearly a twofold increase in surface expression of three CD18-bearing cell surface molecules (CD11a, CD11b, CD11c). The direct involvement of the CD18-bearing molecules in monocyte-mediated cytotoxicity was investigated using monoclonal antibody (MAb) inhibition. MAb recognizing the CD18 subunit significantly inhibited monocyte-mediated killing. The inhibition by anti-CD18 MAb could not be attributed to LFA-1 (CD11a) alone, suggesting that CR3 (CD11b) and p150,95 (CD11c) may also participate in monocyte-mediated cytotoxicity. In contrast, seven of eight other cell surface structures were not affected by PMA treatment, and MAb to all eight cell surface structures did not inhibit killing. These findings suggest that CD18-bearing molecules are upregulated with monocyte activation and may play a functional role in monocyte-mediated killing.     

Three-dimensional architecture of chromosome fibres in the crane fly: co-oriented autosomal bivalents and amphitelic sex univalents during prometaphase

A technique for the fixation of cells during live observation (Nicklas et al. 1979) was used to investigate chromosomes which were moving at the time of fixation. Chromosome fibres were reconstructed by tracking their microtubules in longitudinal serial sections. A considerable proportion of non-kinetochoric microtubules (free microtubules, fMTs) is skewed with respect to the fibre axis. These skew fMTs contribute to the degree of disorder. It was found that the difference in the relative proportion of skew fMTs between active fibres (oriented in the direction of movement) and passive fibres (oriented backwards) is significantly correlated with the chromosome velocity (correlation coefficient r=0.796, P=0.01). It can be concluded that the pulling force generated in the chromosome fibre is a function of skew fMTs.     

Estimation of inter-binding-site distances in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase using Eu(III) luminescence energy transfer

We have used several trivalent lanthanides as probes for the high-affinity Ca(II)-binding site of the Ca(II) + Mg(II)-ATPase of skeletal muscle sarcoplasmic reticulum. The luminescent probes Eu(III) and Tb(III) were excited directly with pulsed laser light and the energy transfer efficiencies to several lanthanide acceptors were measured, under conditions in which most donor-acceptor pair occupied high-affinity Ca(II) sites. We obtain an inter-ionic site distance of about 0.8-0.9 nm. Energy transfer measurements were also done with Eu(III) in at least one Ca(II) site and bidentate Cr-ATP complex at the ATP hydrolytic site. Quenching of Eu(III) luminescence by Cr-ATP was total under these conditions. We calculate an upper limit of 1.0 nm for the distance from the Ca(II) site(s) to the complexed Cr(III) ion at the hydrolytic site.     

Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Inhibition of IL 2-dependent proliferation of rat T lymphoblasts by the monoclonal antibody ART62 which reacts with MHC class 1 antigens

During the course of studies designed to obtain monoclonal antibodies (mAb) that recognize the rat interleukin 2 receptor, a mouse IgG1 mAb (ART62) was identified which inhibits the interleukin 2 (IL 2)-dependent proliferation of rat T lymphoblasts without affecting the binding of IL 2 to such cells. In order to characterize the cell surface components that react with the mAb ART62, T lymphoblasts were surface-labeled with 125I, and the radioactive molecules were immunoprecipitated by the antibody analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mAb ART62 precipitated two major components of 48,000 m.w. and 12,000 m.w., respectively, which were different from those which react with the anti-IL 2-receptor antibody ART18, a molecule of 50,000 to 55,000 m.w. Sequential immunoprecipitation studies revealed that the mAb ART62 reacts with the MHC class 1 antigen that reacts with the classical anti-rat MHC class 1 mAb OX18, and vice versa. In contrast to the mAb ART62, OX18 that does not affect and several other mAbs known to inhibit the rat MLR failed to inhibit IL 2-dependent proliferation of rat T lymphoblasts. In contrast to the anti-IL 2 receptor antibody ART18, ART62 effectively inhibited IL 2-driven proliferation even when added to cells already committed to proliferate by IL 2-IL 2 receptor interaction. These data raise the possibility that MHC class 1 antigens could be involved in the chain of reactions mediating the signals required for cell proliferation.