A novel synthesis of acyclonucleosides via allylation of 3-[1-(phenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one

The allylation of 3-[1-(phenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one (1) gave, in addition to the anticipated 1-N-allyl derivative (2), a dehydrative cyclized product, 1-N-allyl-3-[5-(hydroxymethyl)-1-phenylpyrazol-3-yl]quinoxalin-2-one (4) and its isomeric O-allyl derivative 3. The O-allyl group in 3 underwent acetolysis under acetylation conditions, in addition to the acetylation of the hydroxyl group, to afford 2-acetoxy-3-[5-(acetoxymethyl)-1-phenylpyrazol-3-yl]quinoxaline (8) instead of the O-acetyl derivative of 3. Allylation of the tri-O-acetyl derivative of 1 caused the elimination of a molecule of acetic acid in addition to N-allylation to give 1-N-allyl-3-[3,4-di-O-acetyl-2-deoxy-1-(phenylhydrazono)but-2-en-1-yl]quinoxalin-2-one (11). Hydroxylation of the allyl group gave a glycerol-1-yl acyclonucleoside which can be alternatively obtained by a displacement reaction of the tosyloxy group in 2,3-O-isopropylidene-1-O-(p-tolylsulfonyl)glycerol (14), followed by deisopropylidenation. 1-N-(2,3-Dibromopropyl)-3-[5-(hydroxymethyl)-1-(4-bromophenyl)pyrazol-3-yl]quinoxalin-2-one (15) underwent azidolysis to give a 2,3-diazido derivative. The assigned structures were based on spectral analysis. The activity of compounds 2, 4, 6, and 15 against hepatitis B virus was studied.     

Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.     

The responses of the cabbage seed weevil (Ceutorhynchus assimilis) to the odour of oilseed rape (Brassica napus) and to some volatile isothiocyanates

The responses of the cabbage seed weevil,Ceutorhynchus assimilis Payk. (Coleoptera: Curculionidae) to volatiles from oilseed rape (Brassica napus L.) were tested using a linear track olfactometer. Weevils were attracted towards the odour of rape during a short period before diapause and for most of their postdiapause life. Odours from both the flowering and green parts of the plant were attractive, but the odour of a non-host plant (Bellis perennis L.) was not. An entrainment extract of flowering rape volatiles in pentane was attractive, but significantly less so than the odour of flowering rape itself. Attraction was found to 3-butenyl and 4-pentenyl isothiocyanate (NCS), but not to 2-phenylethyl isothiocyanate. A mixture of the three NCSs was more attractive than the individual NCSs. However, the concentration required to produce a response was still relatively high. Isothiocyanates, along with other volatiles from rape, probably play a role in host plant recognition by the seed weevil.     

In vitro interaction of zajdela ascites hepatoma cells with lipid vesicles

We studied the in vitro interaction between Zajdela ascites hepatoma cells and small unilamellar vesicles, consisting of ¹⁴C-labeled phosphatidylcholine, cholesterol, and phosphatidylserine (molar ratio 5: 4: 1), containing high intravesicular concentrations of carboxyfluorescein or fluorescein isothiocyanate tagged dextran.The entrapped markers were found to be associated with the cells to a lesser degree than the vesicle membrane marker. This discrepancy, which is slightly less pronounced for fluorescein isothiocyanate tagged dextran than for carboxyfluorescein, increases with incubation time and decreases with increasing vesicle lipid concentration in the incubation mixture. Vesicle-plasma membrane exchange of the vesicle lipid marker could not entirely explain the observed discrepancy. It is tentatively concluded that the gap mainly arises from a selective loss of entrapped dyes from vesicles actually interacting with the cell surface. Both spectrofluorimetric and fluorescence microscopic observations, as well as the relative insensitivity of vesicle uptake towards the presence of metabolic inhibitors, exclude a major contribution of endocytosis as a vesicle uptake route. We therefore conclude that vesicles are primarily internalized by a vesicle-cell fusion-like process. The observed discrepancy in uptake between entrapped materials and vesicle lipid is discussed in terms of a two-site vesicle cell surface interaction model.     

A new technique for desorbing substances tightly bound to affinity gels: flat-bed electrophoretic desorption in Sephadex via isoelectric focusing (FEDS-IEF)

In some cases, proteins and other molecules which are tightly bound to affinity gels can be recovered under mild conditions by electrophoresis. We have extended this technique by running electrophoretic desorption in flat-beds of Sephadex in the presence of ampholytes (FEDS-IEF). A number of advantages of this technique are noted: due to the geometry of the apparatus, high voltages can be used which result in short running times; there are no physical barriers to the migration of the protein and no abrupt conductivity drops; desorbed samples are easily located and recovered; and relatively large sample loads can be readily accommodated. Running times are very sensitive to the experimental conditions. Affinity gels should be applied as a narrow zone, distant from the anticipated banding position of the desorbed species. A wide ampholyte interval is generally recommended. The system appears to be gentle and flexible enough to allow investigators to optimize the conditions for desorption of various affinity gel systems.     

Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

The kinetics of renaturation of the β₂-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F₁ and F₂ are studied and compared. Steps corresponding to the refolding of F₁, to the association of the folded F₁ and F₂ fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β₂ starting from the denatured F₁ and F₂ fragments.The step corresponding to the refolding of the F₁ domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β₂-protein. It is concluded that the refolded F₁ fragment corresponds to a folding intermediate on the pathway of renaturation of the β₂-subunit.     

Extracellular space diffusion in central nervous system: anisotropic diffusion measured by elliptical surface photobleaching

Diffusion in the extracellular space (ECS) is crucial for normal central nervous system physiology. The determinants of ECS diffusion include viscous interactions with extracellular matrix/plasma membranes ("viscosity") and ECS geometry ("tortuosity"). To resolve viscosity versus tortuosity effects, we measured direction-dependent (anisotropic) diffusion in ECS in mouse spinal cord by photobleaching using an elliptical spot produced by a cylindrical lens in the excitation path. Anisotropic diffusion slowed fluorescence recovery when the long axis of the ellipse was parallel versus perpendicular to the direction of faster diffusion. A mathematical model was constructed to deduce diffusion coefficients (D(x), D(y)) from fluorescence recovery measured for parallel and perpendicular orientations of the long axis of the ellipse. Elliptical spot photobleaching was validated by photobleaching aqueous-phase fluorophores on a diffraction grating, where diffusion is one-dimensional. Measurement of the diffusion of 70 kDa FITC-dextran in spinal cord in living mice indicated that viscosity slows diffusion by approximately 1.8-fold compared with its diffusion in solution. ECS geometry hinders diffusion across (but not along) axonal fibers in spinal cord further by approximately fivefold. In cerebral cortex, however, approximately 50% of the hindrance to ECS diffusion comes from viscosity and approximately 50% from tortuosity. We suggest that the extracellular matrix might have evolved to facilitate rather than hinder diffusion even for large molecules.     

Folding of beta-sandwich proteins: three-state transition of a fibronectin type III module

An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three-state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two-state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways.     

Beneficial effect of nitric oxide synthase inhibitor on hepatotoxicity induced by allyl alcohol

The effect of aminoguanidine (a selective inhibitor of inducible nitric oxide synthase) on allyl alcohol-induced liver injury was assessed by the measurement of serum ALT and AST activities and histopathological examination. When aminoguanidine (50-300 mg/kg, i.p.) was administered to mice 30 min before a toxic dose of allyl alcohol (75 microL/kg, i.p.), significant changes related to liver injury were observed. In the presence of aminoguanidine the level of ALT and AST enzymes were significantly decreased. All symptoms of liver necrosis produced by allyl alcohol toxicity almost completely disappeared when animals were pretreated with aminoguanidine at 300 mg/kg. Depletion of hepatic glutathione as a consequence of allyl alcohol metabolism was minimal in mice pretreated with aminoguanidine at 300 mg/kg. It was found that the inhibition of toxicity was not due to alteration in allyl alcohol metabolism since aminoguanidine did not effect alcohol dehydrogenase activity both in vivo and in vitro.     

Chemical carboxyl-terminal sequence analysis of peptides and proteins using tribenzylsilyl isothiocyanate

A new derivatization reagent, tribenzylsilyl isothiocyanate (TBS-ITC), is applied to C-terminal peptide and protein sequencing. It has been successfully used to sequence six C-terminal residues of house apomyoglobin and a synthetic peptide at low nanomole levels. The chemistry involves activation with acetic anhydride, derivatization with TBS-ITC, and cleavage of derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. The tribenzylsilyl is a bulky, electric donor group and is a good leaving group. It facilitates the nucleophilic attack of the NCS^–1 in the coupling reaction. The efficiency for C-terminal sequencing by TBS-ITC is about the same as that of acetyl isothiocyanate (AITC), which is a derivatizing reagent for C-terminal sequencing developed by our laboratory. TBS-ITC is much more stable than AITC and trimethylsilyl isothiocyanate (TMS-ITC). TBS-ITC is a solid with relatively long shelf life, whereas AITC and TMS-ITC are liquid and not stable at room temperature.