Comparative studies on the structure and stability of fluorescent proteins EGFP, zFP506, mRFP1, "dimer2", and DsRed1

To obtain more information about the structural properties and conformational stabilities of GFP-like fluorescent proteins, we have undertaken a systematic analysis of series of green and red fluorescent proteins with different association states. The list of studied proteins includes EGFP (green monomer), zFP506 (green tetramer), mRFP1 (red monomer), "dimer2" (red dimer), and DsRed1 (red tetramer). Fluorescent and absorbance parameters, near-UV and visible CD spectra, the accessibility of the chromophores and tryptophans to acrylamide quenching, and the resistance of these proteins to the guanidine hydrochloride unfolding and kinetics of the approaching of the unfolding equilibrium have been compared. Tetrameric zFP506 was shown to be dramatically more stable than the EGFP monomer, assuming that association might contribute to the protein conformational stability. This assumption is most likely valid even though the sequences OF GFP and zPF506 are only approximately 25% identical. Interestingly, red FPs possessed comparable conformational stabilities, where monomeric mRFP1 was the most stable species under the equilibrium conditions, whereas the tetrameric DsRed1 possessed the slowest unfolding kinetics. Furthermore, EGFP is shown to be considerably less stable than mRFP1, whereas tetrameric zFP506 is the most stable species analyzed in this study. This means that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stabilities.     

Interference of calmidazolium with measurement of mitochondrial membrane potential using the tetraphenylphosphonium electrode or the fluorescent probe rhodamine 123

Calmidazolium (CMZ) is a positively charged, hydrophobic compound used as a calmodulin antagonist. It may cause unspecific effects in mitochondria, e.g., a decrease in membrane potential (deltapsi), swelling, and uncoupling. Several groups have advised against use of CMZ in studying signal transduction in mitochondria. We report here that it interferes with measurement of deltapsi in rat liver mitochondria (RLM) when using the tetraphenyl phosphonium (TPP+) electrode. We also found that CMZ reduces the signal, indicating an apparent drop in deltapsi. CMZ itself gave a signal with the TPP+ electrode in the absence of RLM. At high concentrations, > 10 microM, it also reduced the fluorescence quenching of the probe rhodamine 123. This may be due to an interference with mitochondrial uptake and binding of this positively charged probe or to an uncoupling effect. It is concluded that CMZ and similar positively charged calmodulin antagonists such as trifluoperazine may be used in mitochondria if these interferences are controlled and calibration is carried out under the experimental conditions used.     

Thermal and sodium dodecylsulfate induced transitions of streptavidin

The strong specific binding of streptavidin (SA) to biotin is utilized in numerous biotechnological applications. The SA tetramer is also known to exhibit significant stability, even in the presence of sodium dodecylsulfate (SDS). Despite its importance, relatively little is known about the nature of the thermal denaturation pathway for SA. This work uses a homogeneous SA preparation to expand on the data of previous literature reports, leading to the proposal of a model for temperature induced structural changes in SA. Temperature dependent data were obtained by SDS and native polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorescence and ultraviolet (UV)-visible spectroscopy in the presence and absence of SDS. In addition to the development of this model, it is found that the major thermal transition of SA in 1% SDS is reversible. Finally, although SA exhibits significant precipitation at elevated temperatures in aqueous solution, inclusion of SDS acts to prevent SA aggregation.     

HIV-1 Vpu sequesters beta-transducin repeat-containing protein (betaTrCP) in the cytoplasm and provokes the accumulation of beta-catenin and other SCFbetaTrCP substrates

The human immunodeficiency virus type 1 Vpu protein acts as an adaptor for the proteasomal degradation of CD4 by recruiting CD4 and beta-transducin repeat-containing protein (betaTrCP), the receptor component of the multisubunit SCF-betaTrCP E3 ubiquitin ligase complex. We showed that the expression of a Vpu-green fluorescent fusion protein prevented the proteosomal degradation of betaTrCP substrates such as beta-catenin, IkappaBalpha, and ATF4, which are normally directly targeted to the proteasome for degradation. Beta-catenin was translocated into the nucleus, whereas the tumor necrosis factor-induced nuclear translocation of NFkappaB was impaired. Beta-catenin was also up-regulated in cells producing Vpu+ human immunodeficiency virus type 1 but not in cells producing Vpu-deficient viruses. The overexpression of ATF4 also provoked accumulation of beta-catenin, but to a lower level than that resulting from the expression of Vpu. Finally, the expression of Vpu induces the exclusion of betaTrCP from the nucleus. These data suggest that Vpu is a strong competitive inhibitor of betaTrCP that impairs the degradation of SCFbetaTrCP substrates as long as Vpu has an intact phosphorylation motif and can bind to betaTrCP.     

Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan<Emphasis Type="Italic"> Hydra</Emphasis>

To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8–10 m) containing about 3×10⁹ bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5–10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.Abbreviations CT Chromosome territory - BrdU Bromodeoxyuridine - IdU Iododeoxyuridine - CldU Chlorodeoxyuridine Communicated by E.A. Nigg     

The lactose permease of Escherichia coli: overall structure, the sugar-binding site and the alternating access model for transport

Membrane transport proteins transduce free energy stored in electrochemical ion gradients into a concentration gradient and are a major class of membrane proteins, many of which play important roles in human health and disease. Recently, the X-ray structure of the Escherichia coli lactose permease (LacY), an intensively studied member of a large group of related membrane transport proteins, was solved at 3.5 A. LacY is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the molecule. The structure represents the inward-facing conformation, as evidenced by a large internal hydrophilic cavity open to the cytoplasmic side. The structure with a bound lactose homolog reveals the sugar-binding site in the cavity, and a mechanism for translocation across the membrane is proposed in which the sugar-binding site has alternating accessibility to either side of the membrane.     

RNA structure-dependent uncoupling of substrate recognition and cleavage by Escherichia coli ribonuclease III

Members of the ribonuclease III superfamily of double-strand-specific endoribonucleases participate in diverse RNA maturation and decay pathways. Ribonuclease III of the gram-negative bacterium Escherichia coli processes rRNA and mRNA precursors, and its catalytic action can regulate gene expression by controlling mRNA translation and stability. It has been proposed that E.coli RNase III can function in a non-catalytic manner, by binding RNA without cleaving phosphodiesters. However, there has been no direct evidence for this mode of action. We describe here an RNA, derived from the T7 phage R1.1 RNase III substrate, that is resistant to cleavage in vitro by E.coli RNase III but retains comparable binding affinity. R1.1[CL3B] RNA is recognized by RNase III in the same manner as R1.1 RNA, as revealed by the similar inhibitory effects of a specific mutation in both substrates. Structure-probing assays and Mfold analysis indicate that R1.1[CL3B] RNA possesses a bulge– helix–bulge motif in place of the R1.1 asymmetric internal loop. The presence of both bulges is required for uncoupling. The bulge–helix–bulge motif acts as a ‘catalytic’ antideterminant, which is distinct from recognition antideterminants, which inhibit RNase III binding.     

Therapeutic effect of a single peritumoural dose of IL-2 on transplanted murine breast cancer

Interleukin-2 therapy is not clearly effective against breast cancer both in mouse models and in human patients. However, the study of IL-2 therapy of breast cancer remains important, as 3,700 women died from this malignancy in the Netherlands in 2000. Previously we have shown the therapeutical efficacy of a single peritumoural IL-2 application in different experimental models and in veterinary patients. Here we apply this mode of IL-2 therapy to advanced mouse mammary carcinoma models, i.e., severe metastasised tumours in A/Sn mice and non-metastasised carcinomas in BALB/c mice. Mice with advanced transplanted mammary carcinomas were given a single peritumoural treatment with 2.5 x 10(6) IU IL-2 at days 10-14 after i.p. or s.c. inoculation of 10(6) carcinoma cells. Within each experiment it was always possible to distinguish relatively slowly and fast growing tumours which allows the therapeutical effect of IL-2 in tumours with different growth rates to be studied. A new approach to analyse results enabled us to show that survival of mice with transplanted, advanced metastasised breast cancer can be significantly improved after a single local treatment with IL-2. Advanced relatively fast i.p and s.c. growing mammary carcinomas seem to be more sensitive to a single IL-2 treatment than relatively slowly growing tumours. IL-2 was most effective against non-metastasised mouse breast cancer.