Synthesis and characterization of a pegylated derivative of 3-(1,2,3,6-tetrahydro-pyridin-4yl)-1H-indole (IDT199): a high affinity SERT ligand for conjugation to quantum dots

Quantum dots consisting of a cadmium selenide core encapsulated in a shell of cadmium doped zinc sulfide have the potential to revolutionize fluorescent imaging of live cell cultures. In order to utilize these fluorescent probes it is necessary to functionalize them with biologically active ligands. In this paper we report the design and synthesis of a ligand that has a high affinity for the serotonin transporter (SERT) that may be conjugated to quantum dots.     

Adenine-aptamer complexes: a bipartite RNA site that binds the adenine nucleic base.

RNA aptamers that are able to complex free adenine have been isolated by a SELEX (systematic evolution of ligands by exponential enrichment) procedure. The adenine binding site was revealed by sequence alignment for a prevalent cluster of aptamers, and its structure and interactions with adenine were probed by RNase digestion studies, lead cleavage, boundary determination experiments, and truncated sequences studies. A new purine binding motif was functionally and structurally characterized and compared with other RNAs specific to purine or adenylated compounds. The affinity for adenine and the specificity for other related targets were quantified. This work suggests that the adenine binding site is composed of two independent secondary structure elements forming a bipartite binding site that interacts with adenine in a new mode of purine recognition. Such binding is of great interest because the imidazole moiety is not trapped in the binding site, and would easily be available for catalytic activity.     

Rapid Assessment of Microspore and Pollen Development Stage in Wheat and Maize Using Dapi and Membrane Permeabilization

The use of the DNA-specific fluorochrome DAPI has been extended to stage assessment of fresh pollen in wheat and maize. Membrane permeabilization by Triton X-100 incorporated in the staining solution allows access of the fluorochrome to nuclear DNA. At all stages of gametophytic development, the nuclei can be sharply visualized. Starch does not interfere with the fluorochrome so that it is possible to study the second pollen grain mitosis and sperm differentiation. With its rapidity and reliability, this technique represents an efficient tool for routine staging or investigation of the nuclear status of the pollen grains     

Subunit topography of RNA polymerase (E. coli) in the complex with DNA.

E. Coli RNA polymerase binding to different DNAs (from E. Coli, 5-bromodeoxyuridine (BrdUrd) substituted DNA and poly [d(BrU-A)] was induced with ultraviolet (U.V.) light to form protein-DNA crosslinked complexes. Two independent methods of analysis, polyacrylamide gel electrophoresis in SDS and chloroform extraction indicated the formation of a stable complex between the enzyme and DNA. The complexes were formed under different ionic strength conditions, at low enzyme to DNA ratios in order to approach the conditions of specific binding. In contrast there was no crosslinking of the complex in 1 M KCl solution which dissociates the enzyme from DNA. The efficiency of formation of strongly bound complex was found to be much higher with holoenzyme than with core enzyme. The following results were obtained : 1) The large subunits beta and beta' were found to be bound to DNA. 2) Relatively small amount of sigma subunit were bound to DNA while alpha subunits were essentially not attached to DNA. The high binding affinity of beta and beta' subunits was also observed in the studies of isolated subunits. These results lead to a model of enzyme-DNA complex in which the large beta and beta' subunits provide the contacts between the RNA polymerase and the DNA.     

Alteration of the lateral organization of the plasma membrane of Chinese hamster ovary cells by synthetic lipopeptide, Pam3Cys-Ser-Lys4.

The cationic lipohexapeptide (S)-[2, 3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)- Lys 4-OH, trihydrochloride (Pam3Cys-Ser-Lys4) is a synthetic analog of the triacylated N-terminal part of bacterial lipoproteins. In this study we addressed the question of whether Pam3Cys-Ser-Lys4 could modify the organization of the plasma membrane of Chinese hamster ovary cells. 1-Acyl-2-[6-(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C6-NBD-PC) diffusion was followed by fluorescence recovery after photobleaching experiments carried out on the plasma membrane of Chinese hamster ovary cells. Incubation of cells in the presence of Pam3Cys-Ser-Lys4 induced an increase in the lateral diffusion coefficient and in the immobile fraction of C6-NBD-PC probes. Various control experiments have shown that the increase in the immobile fraction was not due to probe internalization induced by Pam3Cys-Ser-Lys4. Back-exchange experiments showed that a good correlation exists between the fractions of immobilized probes and nonextractable probes in the plasma membrane of Chinese hamster ovary cells. A useful way to analyze the origin of probe immobilization (micrometer-sized domains or aggregated patches of proteins) is to carry out fluorescence recovery after photobleaching experiments at variable observation radii. This type of experiment, carried out on the plasma membrane of Chinese hamster ovary cells incubated with Pam3Cys-Ser-Lys4, confirmed that the lipopeptide induced the aggregation of proteins of Chinese hamster ovary plasma membrane. Lipids which were trapped inside these aggregates were thus prevented from diffusing at long range in the plasma membrane plane and behave as an immobile fraction.     

Inhibitory potential of chemical substitutions at bioinspired sites of β-D-galactopyranose on neoglycoprotein/cell surface binding of two classes of medically relevant lectins

Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galectins. Natural anomeric extensions and 3'-substitutions enhance its reactivity, thus prompting us to test the potential of respective chemical substitutions of galactose in the quest to develop potent inhibitors. Biochemical screening of a respective glycoside library with 60 substances in a solid-phase assay was followed by examining the compounds' activity to protect cells from lectin binding. By testing 32 anomeric extensions, 18 compounds with additional 3'-substitution, three lactosides and two Lewis-type trisaccharides rather mild effects compared to the common haptenic inhibitor lactose were detected in both assays. When using trivalent glycoclusters marked enhancements with 6- to 8-fold increases were revealed for the toxin and three of four tested galectins. Since the most potent compound and also 3'-substituted thiogalactosides reduced cell growth of a human tumor line at millimolar concentrations, biocompatible substitutions and scaffolds will be required for further developments. The synthesis of suitable glycoclusters, presenting headgroups which exploit differences in ligand selection in interlectin comparison to reduce cross-reactivity, and the documented strategic combination of initial biochemical screening with cell assays are considered instrumental to advance inhibitor design.     

Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy

The majority of studies on autophagy, a cytoplasmic homeostatis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3‐kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3‐phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI‐1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.     

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages

Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.     

Isolation of two Pseudomonas strains producing pseudomonic acid A

Two novel Pseudomonas strains were isolated from groundwater sediment samples. The strains showed resistance against the antibiotics tetracycline, cephalothin, nisin, vancomycin, nalidixic acid, erythromycin, lincomycin, and penicillin and grew at temperatures between 15 and 37 °C and pH values from 4 to 10 with a maximum at pH 7 to 10. The 16S ribosomal RNA gene sequences and the substrate spectrum of the isolates revealed that the two strains belonged to the Pseudomonas fluorescens group. The supernatants of both strains had an antibiotic effect against Gram-positive bacteria and one Gram-negative strain. The effective substance was produced under standard cultivation conditions without special inducer molecules or special medium composition. The antibiotically active compound was identified as pseudomonic acid A by off-line high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The measurement on ultra performance liquid chromatography (UPLC, UV–vis detection) confirmed the determination of pseudomonic acid A which was produced by both strains at 1.7–3.5 mg/l. Our findings indicate that the ability to produce the antibiotic pseudomonic acid A (Mupirocin) is more spread among the pseudomonads then anticipated from the only producer known so far.     

The role of PI3P phosphatases in the regulation of autophagy

Autophagy initiation is strictly dependent on phosphatidylinositol 3-phosphate (PI3P) synthesis. PI3P production is under tight control of PI3Kinase, hVps34, in complex with Beclin-1. Mammalian cells express several PI3P phosphatases that belong to the myotubularin family. Even though some of them have been linked to serious human diseases, their cellular function is largely unknown. Two recent studies indicate that PI3P metabolism involved in autophagy initiation is further regulated by the PI3P phosphatases Jumpy and MTMR3. Additional pools of PI3P, upstream of mTOR and on the endocytic pathway, may modulate autophagy indirectly, suggesting that other PI3P phosphatases might be involved in this process. This review sums up our knowledge on PI3P phosphatases and discusses the recent progress on their role in autophagy.