Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.     

Comparison of genetic behaviour of the transposable element Tam3 at two unlinked pigment loci in Antirrhinum majus

Summary In Antirrhinum majus the transposable element Tam3 has been described at two unlinked loci pallida and nivea, both of which are required for the production of anthocyanin pigment in flowers. In each case the element is inserted in the promoter region and gives a variegated phenotype. We show that the rate of Tam3 excision at both loci is greatly affected by temperature, being approximately 1000-fold higher at 15°C compared with 25°C. Tam3 is also controlled by an unlinked gene Stabiliser, which considerably reduces excision rate. We show that the high degree of sensitivity to temperature and Stabiliser is an intrinsic property of Tam3 which is not shared by an unrelated element, Tam1. The Tam3 insertion at nivea gives rise to a series of alleles which confer reduced pigmentation, novel spatial patterns and changed instability. These are probably a result of imprecise excision and rearrangements of the Tam3 element.     

Interaction of a fluorescent analogue of GDP with elongation factor Tu: steady-state and time-resolved fluorescence studies

A fluorescent derivative of GDP was prepared by the reaction of 2'-amino-2'-deoxy-GDP with fluorescamine. This derivative binds tightly (KD approximately 4.5 X 10(-8) M) to elongation factor Tu (EF-Tu) from Escherichia coli. The emission properties, including spectra, polarizations, and lifetimes, for fluorescamine-GDP free in solution and bound to EF-Tu are presented. Emission data on the fluorescamine-ethylamine conjugate are also given. A multifrequency phase and modulation lifetime study (using nine modulation frequencies over the range of 2-80 MHz) indicated that the emissions of these three systems were well characterized by single exponential decays corresponding to 1.45 ns for the fluorescamine-ethylamine derivative in buffer and to 7.74 and 11.03 ns for the fluorescamine-GDP derivative free in buffer and bound to EF-Tu, respectively. Multifrequency differential polarized phase fluorometry results indicated a rotational relaxation time of the protein-probe complex of approximately 88 ns; these data also indicated the lack of significant local motion for the probe. Addition of excess GDP to the EF-Tu-probe complex led to displacement of the fluorescamine-GDP derivative as evidenced by the change in both the steady-state and dynamic polarization values. The observed increase in fluorescence intensity upon displacement allowed us to follow the kinetics of the dissociation reaction; a dissociation rate constant of 5.0 X 10(-3) S-1 was determined. These results demonstrate the utility of this 2'-amino-2'-deoxy-GDP analogue as a probe of guanosine nucleotide dependent systems.     

Combination treatment using thymosin α1 and interferon after cyclophosphamide is able to cure Lewis lung carcinoma in mice

Summary A combination treatment with thymosin 1 (200 µg/kg) for 4 days, followed by a single injection of murine interferon / (3 × 10⁴ international units/mouse), starting 2 days after cyclophosphamide treatment (200 mg/kg, single injection) demonstrated a dramatic and rapid disappearance of tumor burden in mice bearing Lewis lung carcinoma (3LL) tumor. The effectiveness of this new chemoimmunotherapy protocol was evident even on the long-term survival in a high percentage of animals, and was statistically significant when compared to treatment with the single agents in conjunction with chemotherapy or to chemotherapy itself. The same combination immunotherapy treatment strongly stimulated natural killer activity and cytotoxicity against autologus 3LL tumor cells in 3LL-tumor-bearing mice treated with cyclophosphamide, whereas treatments with each agent singly did not alter or only slightly modified the cytotoxic activity towards Yac-1 or 3LL target cells. Selective depletion with antibodies showed that killer cells stimulated by combination chemoimmunotherapy treatment bear phenotypic characteristics of asialo-GM1-positive cells. A histological study has shown a high number of infiltrating lymphoid cells in the tumors obtained from mice treated with combination chemoimmunotherapy.     

Anisotropy decay of fluorescence as an experimental approach to protein dynamics

This minireview makes an initial assessment of the progress made using anisotropy decay measurements for investigating the conformational changes and molecular dynamics in soluble systems. A critical analysis of available data is presented. The anisotropy decays of the tryptophan fluorescence of staphylococcal nuclease, adrenocorticotropin, melittin and of labeled transfer RNA were studied for investigating the functional conformational changes of these systems. The emissions of variously labeled immunoglobulins have been used to elucidate the conformations of these proteins before and after the binding of specific antibodies. Labeled myosin and its fragments have given information on the functional motions of the protein domains. The anisotropy decays of labeled and natural hemoglobin systems have been utilized for exploring the allosteric behavior of these molecules. The data suggest a wide applicability of this technique to the study of protein dynamics and conformational changes of macromolecules.     

In vivo electrochemical studies of the effects of cocaine on dopamine nerve terminals in the rat neostriatum

In vivo electrochemical measurements, involving chronoamperometric recordings using monoamine-selective Nafion-coated electrodes, were used to study the effects of locally applied cocaine (50-500 micromolar barrel concentrations) on dopamine (DA) nerve terminals in the neostriatum of the anaesthetized rat. Local application of cocaine did not elicit detectable increases in basal levels of extracellular DA. However, locally applied cocaine significantly augmented the concentration of DA detected following a potassium (K+)-evoked depolarization. Data obtained with a new high-speed chronoamperometric recording technique further support that DA is the predominant species detected electrochemically following potassium-evoked depolarizations both before and after local application of cocaine. Unlike other locally applied uptake inhibitors that we have studied, cocaine failed to augment the time dynamics of released DA. In addition, large doses of the highest concentration of cocaine caused an attenuation of K+-evoked DA release, presumably due to cocaine's local anaesthetic properties. These data suggest that cocaine elevates synaptic levels of DA, but in a manner that is not identical to other potent monoamine uptake inhibitors.     

Increased membrane heterogeneity in stimulated human granulocytes

TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes and redistribution of membrane components.     

Species dispersion profiles of anthropogenic grasslands in the Italian Eastern Pre-Alps

A method is proposed to analyse the dispersion profiles of species in classes of environmental variables, based on the decomposition of the expected frequencies in contingency tables with many interacting species. The method has been applied to data of dominant or very frequent graminoid species in grasslands of the Natisone Valley (Friuli, Italy). It allowed to make predictions by removing the random component of variation.Nomenclature follows: P. Zangheri (1976). Flora Italica, CEDAM, Padua.The authors were recipients of an Italian CNR grant (E. Feoli) and a Canadian NSERC grant (L. Orlóci) during tenure of this project. The results are in partial fulfillment of a commitment to the Centro Regionale per la Sperimentazione Agraria per il Friuli-Venezia Giulia.