Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer

Chimeric -glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BrdUrd bromodeoxyuridine - GUS -glucuronidase - Km^R kanamycin resistant - T-DNA transferred DNA     

Efficient orthogonal bioconjugation of dendrimers for synthesis of bioactive nanoparticles

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.     

How Does Circadian Rhythm Impact Salt Sensitivity of Blood Pressure in Mice? A Study in Two Close C57Bl/6 Substrains

BackgroundMouse transgenesis has provided the unique opportunity to investigate mechanisms underlying sodium kidney reabsorption as well as end organ damage. However, understanding mouse background and the experimental conditions effects on phenotypic readouts of engineered mouse lines such as blood pressure presents a challenge. Despite the ability to generate high sodium and chloride plasma levels during high-salt diet, observed changes in blood pressure are not consistent between wild-type background strains and studies.MethodsThe present work was designed in an attempt to determine guidelines in the field of salt-induced hypertension by recording continuously blood pressure by telemetry in mice submitted to different sodium and potassium loaded diets and changing experimental conditions in both C57BL/6N and C57BL/6J mice strain (Normal salt vs. Low salt vs. High-salt/normal potassium vs. High salt/low potassium, standard vs. modified light cycle, Non-invasive tail cuff blood pressure vs. telemetry).ResultsIn this study, we have shown that, despite a strong blood pressure (BP) basal difference between C57BL/6N and C57BL/6J mice, High salt/normal potassium diet increases BP and heart rate during the active phase only (dark period) in the same extent in both strains. On the other hand, while potassium level has no effect on salt-induced hypertension in C57BL/6N mice, high-salt/low potassium diet amplifies the effect of the high-salt challenge only in C57BL/6J mice. Indeed, in this condition, salt-induced hypertension can also be detected during light period even though this BP increase is lower compared to the one occurring during the dark period. Finally, from a methodological perspective, light cycle inversion has no effect on this circadian BP phenotype and tail-cuff method is less sensitive than telemetry to detect BP phenotypes due to salt challenges.ConclusionsTherefore, to carry investigations on salt-induced hypertension in mice, chronic telemetry and studies in the active phase are essential prerequisites.     

Regulation of Flagellar Morphogenesis by Temperature: Involvement of the Bacterial Cell Surface in the Synthesis of Flagellin and the Flagellum

The induction of beta-glucosidases (EC 3.2.1.21) was studied in Neurospora crassa. Cellobiase was induced by cellobiose, but other inducers had little effect on this enzyme. Cellobiase activity was very low in all stages of the vegetative life cycle in the absence of di-beta-glucoside inducer. Aryl-beta-glucosidase was semiconstitutive at late stages of culture growth prior to conidiation. At early stages, aryl-beta-glucosidase was induced by cellobiose, laminaribiose, and gentiobiose, and weakly induced by galactose, amino sugars, and aryl-beta-glucosides. The induction properties of the beta-glucosidases are compared with those of the other disaccharidases of Neurospora. The induction of beta-glucosidases was inhibited by glucose, 2-deoxy-d-glucose, and sodium acetate. Sodium phosphate concentrations between 0.01 and 0.1 M stimulated induction of both enzymes, while concentrations above 0.1 M were inhibitory. The optimal condition for induction of both beta-glucosidases was pH 6.0. Cellobiase induction was relatively more inhibited than aryl-beta-glucosidase in the range of pH 6.0 to 8.0.     

Effect of taurine,l-glutamine and l-histidine addition in an amino acid glucose solution on the cellular bioavailability of zinc

Radioactive zinc was used to study the effect of a binary parenteral nutrient solution, composed of amino acids and glucose, on zinc uptake by fibroblasts. The influence of addition of taurine, l-glutamine and of the increase in l-histidine content of the admixture was assessed. The pure mixture was highly toxic for cells and so it was diluted 1/5 in tyrode buffer with 2% albumin. As compared with cells incubated in the buffer containing albumin, zinc absorption was significantly higher (P < 0.05) in the presence of the amino acids of the mixture. Amino acids thus increased bioavailability by displacing zinc bound to albumin. When the histidine concentration in the nutrient medium (4.2 mm) was doubled, inhibition was noted after 30 min of incubation and zinc uptake thereafter remained comparable to that in histidine-free medium. The addition of glutamine (4.2 mm), usually not present in binary mixtures, resulted in significant differences as compared with glutamine-free control medium. Taurine (0.8 mm), led to a constant increase in zinc uptake by fibroblasts as compared with that obtained with taurine-free mixture. However, ultrafiltration showed that taurine was not able to displace zinc from albumin.     

Photoaffinity labeling of scorpion toxin receptors associated with insect synaptosomal Na+ channels

Photoreactive and radioiodinated derivatives of several scorpion toxins acting on insect Na+ channels were prepared without loss of their pharmacological activities. Photoaffinity experiments were carried out on a synaptosomal fraction from the nerve cord of the cockroach Periplaneta americana: with all toxin derivatives, a single specifically labeled band was obtained with a molecular weight of 188,000 +/- 12,000 (n = 17). These results indicate for the first time the molecular weight of the scorpion toxin receptor from the insect nervous system which is probably associated with voltage sensitive Na+ channels. One of these toxins, toxin VII from Tityus serrulatus venom, has been previously shown to be active both in mammals and in insects, in rat brain synaptosomes this toxin labeled a Mr = 31,000 +/- 4,000 band in contrast, to observations in the insect preparation.     

Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.