A simple and efficient synthesis of puromycin, 2,2′-anhydro-pyrimidine nucleosides,cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl Xylo-nucleosides

Synthesis of antibiotics, puromycin and 3 ′-amino-3 ′-deoxy-N ⁶,N ⁶-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3 ′,5 ′-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-α-carbamate 7. In a similar manner, 2,2 ′-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2 ′,3 ′-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.     

A novel computational framework for D(t) from Fluorescence Recovery after Photobleaching data reveals various anomalous diffusion types in live cell membranes

Diffusion of proteins and lipids in lipid membranes plays a pivotal role in almost all aspects of cellular biology, including motility, exo?/endocytosis and signal transduction. For this reason, gaining a detailed understanding of membrane structure and function has long been a major area of cell biology research. To better elucidate this structure‐function relationship, various tools have been developed for diffusion measurements, including Fluorescence Recovery After Photobleaching (FRAP). Because of the complexity of cellular microenvironments, biological diffusion is often correlated over time and described by a time‐dependent diffusion coefficient, D(t) , although the underlying mechanisms are not fully understood. Since D(t) provides important information regarding cellular structures, such as the existence of subresolution barriers to diffusion, many efforts have been made to quantify D(t) by FRAP assuming a single power law, D(t) = Γt ^{α ? 1} where Γ and α are transport coefficient and anomalous exponent. However, straightforward approaches to quantify a general form of D(t) are lacking. In this study, we develop a novel mathematical and computational framework to compute the mean square displacement of diffusing molecules and diffusion coefficient D(t) from each individual time point of confocal FRAP data without the single power law assumption. Additionally, we developed an auxiliary equation for D(t) which can readily distinguish normal diffusion or single power law anomalous diffusion from other types of anomalous diffusion directly from FRAP data. Importantly, by applying this approach to FRAP data from a variety of membrane markers, we demonstrate the single power law anomalous diffusion assumption is not sufficient to describe various types of D(t) of membrane proteins. Lastly, we discuss how our new approaches can be applied to other fluorescence microscopy tools such as Fluorescence Correlation Spectroscopy (FCS) and Single Particle Tracking (SPT).     

Selective Biocatalytic Acylation Studies on 5′-O-(4,4′-Dimethoxytrityl)-2′,3′-Secouridine: An Efficient Synthesis of UNA Monomer

Lipozyme® TL IM (Theremomyces lanuginosus lipase immobilized on silica) in toluene catalyzes the acylation of the 2 ′-OH over the 3 ′-OH group in 5 ′-O-(4,4 ′-dimethoxytrityl)-2 ′,3 ′-secouridine (5 ′-O-DMT-2 ′,3 ′-secouridine) in a highly selective fashion in moderate to almost quantitative yields. The turn over during benzoyl transfer reactions mediated by vinyl benzoate or benzoic anhydride was faster than in acyl transfer reactions with vinyl acetate or C₁ to C₅ acid anhydrides; except in the case of butanoic anhydride. The 2 ′-O-benzoyl-5 ′-O-DMT-2 ′,3 ′-secouridine obtained by Lipozyme® TL IM catalyzed benzoylation of 5 ′-O-DMT-2 ′,3 ′-secouridine was successfully converted into its 3 ′-O-phosphoramidite derivative in satisfactory yield, which is a building block for the preparation of oligonucleotides containing the uracil monomer of UNA (unlocked nucleic acid).     

The Presence of Novel Plant Growth Regulators in Leaves of Distylium racemosum Sieb et Zucc

The scaling law derived from the percolation theory was applied to the concentration dependence of mechanical properties of polyacrylamide measured near the sol–gel transition point. The critical concentration of the sol–gel transition, ?_g, was estimated from the plot of concentration (?) vs. the reciprocal of viscosity (η) by extrapolating 1/η to zero. The critical exponent for the sol viscosity, s, which was estimated from the slope of the log(?_g–?) vs. log η plot was about 0.7. The estimated value of s was similar to the value predicted by the percolation theory based on the superconductor–normal conductor mixture model. The critical exponent for the gel elasticity, t, as estimated from the slope of the log(?–?_g) vs. log G′ plot, where G′ was the dynamic shear modulus of the gel at a frequency of 2Hz. The value of t was about 2, which was also similar to the value predicted by the percolation theory. These results indicated the at the concentration dependences of η and G′ of polyacrylamide near the sol–gel transition point were described by the percolation theory.     

Computer Simulation of Vapor-Liquid Phase Separation

Abstract

A late-time growth law of domains undergoing vapor-liquid phase separation is studied for two- and three-dimensional Lennard-Jones fluids by molecular dynamics simulations. The characteristic domain size shows a power law growth in a late stage with the growth exponent of ½ for both two- and three-dimensional fluids. This study concerns also the relationship between statistical properties of domain patterns and temperatures. The asymptotic form factor of each system is obtained using scaling and the asymptotic tail of the form factor is analyzed. This tail is related to the domain-wall structure. At low system temperatures, the form factor satisfies Porod's law; the asymptotic tail decreases as S(k) ～ k ^{?(D+ 1)} where D is the system dimensionality. However, it is found that the decay of the asymptotic tail becomes slower than that of the Porod tail at higher temperatures in both two- and three-dimensional systems. This indicates that the dimension of the domain wall is fractal and increases with increasing system temperature.     

Synthesis and Biological Evaluation of (3′S)-3′-Deoxy-3′-Fluoro-3′-C-Ethynylcytidine

The novel pyrimidine nucleoside, (3 ′S)-3 ′-deoxy-3 ′-fluoro-3 ′-C-ethynylcytidine (1) was synthesized from cytidine in seven steps. The key step in the synthesis was the introduction of the tertiary fluorine at the 3 ′-position. Compound 1 was evaluated in vitro against several RNA viruses.     

The aggregation of reverse micelles

We have developed a computer simulation to model the formation of reverse micelles in two dimensions. Several of the qualitative results obtained from these calculations agree with experimental observations. Specifically, we have shown that the chain length has a large influence in determining the size and shape of the aggregate. We predict the existence of a critical tail length: Chains below this value will form an extended lamella-like structure, whereas chains longer than this value will form clusters that appear ellipsoid or circular in cross-section. Finally, we obtained a scaling law that relates the aggregation number (N) to the length of the tail (L):N∼L ^−1.14. A physical model to account for the observed exponent will be developed in a future paper.     

Scaling of standard metabolic rate in estuarine crocodiles Crocodylus porosus

Standard metabolic rate (SMR, ml O₂ min^?1) of captive Crocodylus porosus at 30 °C scales with body mass (kg) according to the equation, SMR = 1.01 M^0.829, in animals ranging in body mass of 3.3 orders of magnitude (0.19–389 kg). The exponent is significantly higher than 0.75, so does not conform to quarter-power scaling theory, but rather is likely an emergent property with no single explanation. SMR at 1 kg body mass is similar to the literature for C. porosus and for alligators. The high exponent is not related to feeding, growth, or obesity of captive animals. The log-transformed data appear slightly curved, mainly because SMR is somewhat low in many of the largest animals (291–389 kg). A 3-parameter model is scarcely different from the linear one, but reveals a declining exponent between 0.862 and 0.798. A non-linear model on arithmetic axes overestimates SMR in 70 % of the smallest animals and does not satisfactorily represent the data.     

Electrochemical Analysis of 3′-Azidothymidine (AZT)

Abstract

3′-Azido-3′-deoxythymidine (AZT) exhibits a two-electron diffusion—controlled polarographic reduction wave, with conversion to 3^′?amino-3′-deoxythymidine. The mechanism of reduction, analytical and clinical applications, and its use for one-step synthesis of amino from azido nucleosides, are described.     

Haber's Rule: The Search for Quantitative Relationships in Toxicology

Dose (or concentration) and time of exposure are both important in determining the intensity of response to a toxic agent. For a given response intensity, Haber's Rule (c×t=K) has been proposed as a law of toxicology, but this rule is just one special case of a more general relationship c×t ^m =K, where the exponent m is quite variable. For inhaled toxicants m generally has a value between 0 and 1, whereas for carcinogens m is usually between 1 and 5. The absence of a universal value for m, or one that is generally applicable to different classes of toxicants, makes it not yet possible to develop a Haber-type rule with which to extrapolate successfully between exposure scenarios.     

Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET2

Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET² pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP². This BRET² sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET² sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (G_αs) and the polycationic compound polylysine. G_αs, but not the CK2 substrate β-arrestin2, decreased the BRET² signal by up to 50 %. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50 %. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET² sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.     

Memory effects on scale-free dynamics in foraging Drosophila

The fruit fly, Drosophila melanogaster, displays a scale-free behavior in foraging, i.e., the dwell time on food exhibits a power law distribution. The scaling exponent is generally believed to be stable and the significance of the exponent itself with respect to the scale-free behavior remains elusive. We propose a model whereby the scaling exponent of the scale-free behavior of an animal depends on the memory of the individual. The proposed model is based on the premise that animal behaviors are associated with internal states of the animal. The changes in the scaling exponent are derived by considering losing memory as increasing uncertainty, which is expressed in terms of information entropy of the internal states. Predicted model behaviors agree with experimental results of foraging behavior in wild-type and learning/memory Drosophila mutants. The concept of changes in the scaling exponent due to the amount of memory provides a novel insight into the emergence of a scale-free behavior and the meaning of the scaling exponent.     

Reproductive biology of the invasive Asian freshwater leech Barbronia weberi (Blanchard, 1897)

Barbronia weberi were collected from Hydrilla verticillata purchased from aquarium suppliers in Melbourne, Australia and additional B. weberi were obtained from aquatic plants in Canberra, ACT, Australia. Adult leeches fed Tubifex sp. ad libitum successfully reproduced under laboratory conditions (21 ± 5?°C ). Reproductively mature B. weberi produced cocoons every week for up to three months at which time the adults began to senesce. Cocoons contained one to five (barx=2.41±0.78 SD, N=58) eggs with juveniles leaving the cocoon within 27 ± 3.4 SD days (N=13) of cocoon production. Juvenile B. weberi reached reproductive maturity within four months (N=7) of hatching and had a maximum growth rate of 1.10 mm² d^?1 once they left the cocoon at around 30 days. Individual egg volume (r=0.63, scaling exponent = 1.21) and cocoon volume (r=0.65, scaling exponent = 1.24) showed a significant (P<0.001) and nearly isometric relationship when scaled with maternal body size. Because this species can grow rapidly and produce a large number of eggs over a short period of time and can piggyback with plant species and travel through the aquarium trade, there is potential for B. weberi to rapidly invade new localities.     

Transient electric birefringence study of highly dilute agarose solutions

In order to characterize the first step of agarose gelation, highly dilute solutions (2·10^?3 to 0.5 g/L) have been studied by means of the transient electric birefringence technique. The field-free decay curves of the birefringence are well described by a stretched-exponential B(t) ≈ exp(?t/τ)^β; the value of the exponent β is close to 0.5 whatever the agarose concentration. The suspended particles observed by electron microscopy present a rod-like shape with a constant diameter (～50 Å), without any branching; they are polydisperse with a distribution of lengths approximately exponential. The mean length of these fibers, deduced from their mean rotational diffusion coefficient, is proportional to the 0.37 power of the agarose concentration in the solution. Furthermore, these particles possess a strong permanent electrical dipole confirming the side-to-side arrangement of helices into bundles; this dipole is roughly proportional to the particle length, indicating a self-similarity in the unidirectional growth of the agarose fibers, even when approaching the gelling concentration.     

DDX Profiles in Agricultural Fields Used for Cucurbit Production in Sakarya,Turkey

DDT (2,2-bis(chlorophenyl)-1,1,1- trichloroethane) and its metabolites DDD (2,2-bis(chlorophenyl)-1,1 -dichloroethane) and DDE (2,2-bis(chloraphenyl)-1,1 –dichloroethylene) have half-lives in soil measured in years or decades and are classified as Persistent Organic Pollutants (POPs). In this study, p,p′-DDT, p,p′-DDD, and p,p′-DDE residues were investigated in select agricultural fields of Sakarya Province, Turkey, where Cucurbitaceae have been grown for many years. Total squash and pumpkin production in Sakarya is approximately 3% of total cucurbit production of Turkey but little is known about the concentrations of DDT, DDD, and DDE in these agricultural soils. Thirty-three soil samples were collected from agricultural fields in different counties of Sakarya. p,p′-DDT was detected in all soil samples, with concentrations ranging from 0.23 ng/g to 123 ng/g soil (dry weight). The concentrations of p,p′-DDT metabolites ranged from nondetectable (<0.06 ng/g) to 120 ng/g for p,p′-DDD and from nondetectable (<0.03 ng/g) to 294 ng/g for p,p′-DDE. The highest total DDX (sum of p,p′-DDT, p,p′-DDD, and p,p′-DDE) concentrations among the soil samples was 428 ng/g in a sample collected from Karasu County. Further research in this field was conducted to measure p,p′-DDT, p,p′-DDE, and p,p′-DDD concentrations at multiple locations as a function of soil depth. p,p′-DDT concentrations were measured from 52 ng/g to 1935 ng/g at 0–60 cm depth. The highest DDX concentration was observed at a location where plants have been actively grown since 1987. The lowest DDX concentrations were observed where crops have not been grown since 1987. Our data proved that soil DDX levels at the field gradually increased as a function of how extensively the field has been used for cucurbits production. However, it is not certain whether the application of p,p′-DDT was terminated or if there may still be illegal usage in agricultural soils.     

Kinetics of partly diffusion controlled reactions XXIV: Unidimensionalization of diffusion in cylindrical symmetry systems: Applications to membranes

We show that it is possible to change the space of diffusion to determine the diffusion controlled apparent rate constant of reaction k _a (t). The most important result is that k _a (t) can be always expressed by a simple law which will be used for reactions in an infinite plane to an infinite 3D spherical space.     

Convenient Synthesis of 2′-Deoxy-2-fluoroadenosine from 2-Fluoroadenine

Abstract

A convenient synthesis of 2′-deoxy-2-fluoroadenosine from commercially available 2-fluoroadenine is described. The coupling reaction of silylated 2-fluoroadenine with phenyl 3,5-bis[O-(t-butyldimethylsilyl)]-2-deoxy-1-thio-D-erythro-pentofuranoside gave the corresponding 2-fluoro-2′-deoxyadenosine derivative (α/β =1:1) in good yield. The α- and β-anomers were separated by chromatography, and then desilylated to give compounds 1a and 1b.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Daily rhythms after vaccination on specific and non-specific responses in Nile tilapia (Oreochromis niloticus)

ABSTRACT

We evaluated the daily changes in immunological and hematological factors in tilapia (Oreochromis niloticus) after an immunization period with a subsequent challenge. Experiments were divided into two phases: Phase 1 (immunization): 144 fish were distributed into two groups with 72 fish in six tanks. One group (T1) was immunized, comprising six vaccination time points (ZT schedule = ZT2 h, ZT6 h, ZT10 h, ZT14 h, ZT18 h, ZT22 h). The same schedule was applied to the other group, but with saline solution (non-vaccinated: T2). Both groups remained in the laboratory for 30 days (considered the immunization period). Phase 2 (challenge): on day 30, both vaccinated and non-vaccinated groups were challenged with Streptococcus agalactiae (2.0 × 10⁷ CFU mL^?1) following the same ZT schedule to stimulate the immune response without leading to widespread infection and mortality. On day 45, blood and head kidney samples were collected during the same ZT schedule. The variations in time of the following parameters within each group were evaluated: hematology, peroxidase activity, IgM, tnf-α3, tgf-β1, il-1β and il-12 gene expression. No significant mortality was observed for the groups or the ZT schedule (p > 0.05). Daily rhythms with diurnal acrophases were found in T2 for il12, tnf-α3 and tgf-β1 expression gene, while the acrophases of the peroxidase level, hematocrit and thrombocytes were at nighttime (p < 0.05). In contrast, most of the parameters in the vaccinated tilapia showed no daily rhythms (p > 0.05), except IgM. For all the parameters, the interaction effect between time and treatment (vaccinated and non-vaccinated groups) depended on ZT. Our results reveal that the humoral and non-specific immune system displayed a circadian rhythm based on the light-dark cycle, which could be affected by the vaccination procedure in tilapia.     

Combining particle tracking microrheology and viscometry for the study of DNA aqueous solutions

We use video particle tracking microrheology (VPTMR) in order to investigate the viscoelasticity of salmon DNA and correlate it to its steady-flow shear-thinning viscosity. Aqueous solutions of DNA are tested in a wide concentration range from the dilute to the semidilute unentangled concentration regime. The observed mean squared displacement shows power-law scaling with lag-time which is equivalent to power-law behavior of the complex modulus as a function of frequency that is, |G^*(ω)| = S ∙ ω ^α . The relaxation exponent α changes abruptly with concentration in the semidilute regime from about 1 to about 0.5 which is the exponent predicted by the Rouse model. The quasi-property S follows the scaling of viscosity for uncharged polymers near θ-conditions in the semidilute regime that is, with ν_eff = 0.50 − 0.51 . The shear-thinning exponent observed by viscometry increases gradually towards the value of 0.5 which has been predicted for Rouse chains under flow. Our findings are in agreement with recent studies of DNA solutions where DNA is treated as a model polymer and addresses the low-molar mass regime of DNA viscoelasticity. This work demonstrates that the combination of passive particle tracking with viscometry can provide a complete picture on the viscoelasticity of DNA-based biopolymer materials.