Synthesis of mitochondrial DNA in spermatocytes of Rhynchosciara hollaenderi

During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm³; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 10⁶. Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 10⁵ daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.     

Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

On the molecular length of the replicative form DNA of bacteriophage phiX174

This paper describes an electron microscopic study of the circular replicative form DNA of bacteriophage φX174. The study has been carried out using a preparative technique in which the DNA molecules are adsorbed from solution on to the cleavage surface of mica and visualized in the electron microscope as a metal-shadowed replica (Gordon &; Kleinschmidt, 1969,1970). Contour lengths of open circular molecules were measured in samples obtained from preparations in which the following experimental parameters were varied: the ionic strength of the solution from which the DNA was adsorbed on the mica and the way in which the molecules were dried before shadowing. At the 0.05 significance level, varying these parameters had no effect on the mean length and variances of samples of molecules obtained from five experiments; the samples were therefore regarded as being drawn from the same molecular population with a mean length and variance of, respectively, 1.83 μm and 0.0117 μm².It was argued that the DNA molecules adsorbed on the mica are “frozen” into the molecular conformation present in solution at the time of adsorption and that, therefore, the experimentally determined contour lengths represent authentic molecular lengths in solution. Based on current estimates of the replicative form DNA molecular weight, the mean contour length obtained was slightly but significantly larger than the length predicted for molecules in an exact B configuration. The variance was larger than could be attributed solely to experimental error, indicating that the molecular population in aqueous solution is heterogeneous in contour length. These experimental results were shown to be consistent with a model for DNA structure in aqueous solution in which individual molecules are dynamic variants of a perturbed B form structure (von Hippel &; Wong, 1971).     

Growth, membrane potential and endogenous ion currents of willow (Salix viminalis) roots are all affected by abscisic acid and spermine

Growth measurements of hormone-treated roots from willow cuttings were combined with electrophysiological recordings to study hormone-induced changes in membrane potential and in endogenous ion currents. The mean growth rate of roots was 10 ± 2 μm min^?1 in regular nutrient solution. It increased to 13 ± 2 μm min⁺¹ after application of spermine and decreased to 0.07 ± 0.01 μm min^?1 after treatment with abscisic acid (ABA). Transient depolarizations were elicited in root cortex cells by spermine, while ABA caused a transient hyperpolarization. All changes in membrane potential were accompanied by transient responses of the endogenous current. These responses suggest that first anions, then cations leave the root during spermine-induced depolarizations. From the changes of the endogenous current an apparent efflux of anions (presumably Cl^?) and cations (presumably K⁺) of 200 to 700 pmol cm^?2 per depolarization was calculated. To further investigate a possible relation between endogenous ion currents, growth and the growth regulators ABA and spermine, long-lasting extracellular vibrating-probe measurements were performed. Control roots showed an inward current of about 1.5 μA cm^?2 at the apical elongation zone and an outward current with a maximum density of 1.3 μA cm^?2 at the central and basal elongation zone. The addition of ABA and spermine (final concentration 0.1 mM) to the bathing medium affected the endogenous current in opposite ways: ABA caused a reduction of inward and outward current, while spermine stimulated both. Since protons are a major component of the endogenous current, and sucrose can be taken up by root cells from the apoplast via symport with H⁺, a role of the endogenous current in growth regulation is indicated.     

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/?IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/?IRE construct. ⁶⁴Cu¹⁺ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. ⁶⁴Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein^?1 min^?1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/?IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu¹⁺.     

Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments

We have examined the structure of actin-binding molecules in solution and interacting with actin filaments. At physiological ionic strength, actin-binding protein has a M_r value of 540 × 10³ as determined by direct and indirect hydrodynamic measurements. It is an asymmetrical dimer composed of 270 × 10³ dalton subunits. Viewed in the electron microscope after negative staining or low angle shadowing, actin-binding protein molecules assume a broad range of conformations varying from closed circular structures to fully extended strands 162 nm in contour length. All configurations are apparently derived from the same structure which consists of two monomer chains connected end-to-end. The radius of gyration determined from the electron microscopic images was 21.3 nm in agreement with the value of 17.6 nm calculated from hydrodynamic assays. The average axial ratio from hydrodynamic measurements was 17:1, whereas fully extended dimer molecules in the electron microscope would have an axial ratio of 54:1. All of these observations indicate that actin-binding protein dimers are extremely flexible. The flexibility parameter λ (Landau &; Lifshits, 1958) for actinbinding protein is 0.18 nm^?1.As determined by sedimentation, actin-binding protein binds to actin filaments with a K_a value of 2 × 10⁶m^?1 and a capacity of one dimer to 14 actin monomers in filaments. After incubation of high concentrations (molar ratio to actin ≥ 1:10) of actin-binding protein with actin filaments, long filament bundles are visible in the electron microscope. Under these conditions, actin-binding protein molecules decorate the actin filaments in the bundles at regular 40 nm intervals or once every 15 monomers, approximately equivalent to the binding capacity measured by sedimentation. Low concentrations of actin-binding protein (molar ratio to actin ≥ 1:50) which promote the gelation of actin filaments in solution, did not detectably alter the isotropy of the actin filaments. Direct visualization of actinbinding protein molecules between actin filaments in the electron microscope showed that dimers are sufficient for crossbridging of actin filaments and that actinbinding protein dimers are bipolar, composed of monomers connected head-to-head and having actin-binding sites located on the free tails.We conclude that actin-binding protein is a dimer at physiological ionic strength. Each dimer has two actin filament binding sites and is therefore sufficient to gel actin filaments in solution. The length and flexibility of the actin-binding protein subunits render this molecule structurally suited for the crosslinking of large helical filaments into isotropic networks.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Breakage by hydrodynamic shear of the bonds between cohered ends of lambda-DNA molecules

The rate of breakage by hydrodynamic shear of the cohered ends of λ-DNA molecules has been observed for the circular monomers, joined half molecules, and joined quarter molecules, in a capillary apparatus with known flow parameters. The rate constant for breakage has been measured as a function of shear stress, temperature, ionic strength, and molecular length. There is a large temperature coefficient, with an activation energy of 120 ± 20 kcal./mole. The values of d ln k/dG, where k is the rate constant for breaking and G is shear gradient, in aqueous solution at 25°C. are about 3.8 ± 0.3 × 10^?4 see. The shear stresses needed for breakage of joined quarter molecules and of circular monomers, respectively, are about equal, and about half that needed for breakage of joined half molecules. The rate of breakage at a given shear stress increases with decreasing ionic strength, approximately as [Na⁺]^?1.6. Self-protection effects are not observed for opening of circular monomers at a DNA concentration of 5 μg./ml. but are observed for breakage of joined half molecules at concentrations down to 0.5 μg./ml. The large temperature coefficient which is approximately equal to that of the thermal dissociation of the cohered ends is interpreted to mean that shear breakage is a mechanically assisted thermal reaction in which the thermal fluctuations provide most of the free energy of activation for breakage. A detailed model for this interpretation is presented. The self-protection effect implies that those molecules which break are not average molecules but exceptional ones which, due to some fluctuation, are more fully extended in the flow field.     

Displacement and Return Movement of Chloroplasts in the Marine Dinophyte Pyrocystis noctiluca. Experiments with Optical Tweezers

Infrared laser traps (optical tweezers) were used to study laser-induced organelle movements in the marine alga Pyrocystis noctiluca (Dinophyta). These cells are highly suitable for optical micromanipulation due to their large size and extensive vacuole. Experiments were done with plastids held by optical tweezers and moved from the nuclear area into the vacuole. The subsequent retraction movement was analysed for speed. The displaced organelles remained connected to their original position by a thin cytoplasmic strand, often less than 1 μm in diameter. When the organelles were released they rapidly returned at an initial rate of 81.7 ± 7.8 μm . s^?1 (overall displacement 50 μm, measured distance 20 μm, 25 °C ± 1 °C, number of cells 22), slowing down with progressive retraction of the connecting strand. The return movement was reduced to 4.2 ± 0.2 μ .s^?1 (n = 10) when the organelles were displaced and held for 1 min. Displacement to a longer distance increased the rate of return movement. A change from a high to a low environmental temperature significantly reduced movement from 94.5 ± 9.0 . s^?1 (30 °C ± 1 °C, n = 22) to 34.5 ± 2.7 μm .s^?1 (5°C ± 1 °C, n = 22). Nocodazole and N-ethylmaleimide (NEM), inhibitors of microtubules and acto-myosin, respectively, did not affect the retraction of the connecting strand, but at high concentrations of NEM it became increasingly difficult to move organelles away from the nuclear area. We suggest that the return movement of organelles within laser-induced artificial strands mainly depends on the viscoelastic properties of the tonoplast. The quantification of these properties by optical tweezers allows determination of reactions of plant cells to temperature changes.     

Regulation of catecholamine synthesis in rat brain synaptosomes

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent K_m of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10^?6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10^?7m ; noradrenaline, 25% inhibition at 5 ± 10^?6m ;and serotonin, 30% inhibition at 10^?5m . The catecholamine-induced inhibition of synthesis was antagonized by pre-incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra-synaptosomal amine levels, and that short-term exposure to depolarizing concentrations of potassium can increase synthesis.     

PHOSPHORUS AND NITROGEN NUTRITION IN CHONDRUS CRISPUS (RHODOPHYTA): EFFECTS ON TOTAL PHOSPHORUS AND NITROGEN CONTENT,CARRAGEENAN PRODUCTION,AND PHOTOSYNTHETIC PIGMENTS AND METABOLISM1

The existence of a phenomenon in phosphorus (P) nutrition comparable to the “Neish effect” in nitrogen (N) nutrition (an inverse relation between seawater N enrichment and carrageenan content) was investigated in the temperate red alga Chondrus crispus Stackhouse. Plants were preconditioned for 17 d and then cultured under varying enrichments of P (0, 3, 6, 10, 15 μM P·wk^?1) and a constant N enrichment (53.5 μM N·wk^?1) for 5 wk. Tissue total P, tissue total N, and carrageenan contents were then determined. Identical experiments were performed using C. crispus collected during the fall, winter, spring, and summer seasons. The procedure was repeated using material collected during the following fall season and cultured under constant P (6 μM P·wk^?1) and varying N enrichments (0, 3, 6, 10, 25 μM N·wk^?1). In the fall (P) experiment, carrageenan content was the highest [53.1 ± 0.3% DW (dry weight)], and tissue total P content was the lowest (1.71 ± 0.27 mg P·g DW^?1) in plants that received no P enrichment. Carrageenan content was stable (46.1 ± 1.8% DW) for plants given enrichments of 3 μM P·wk^?1 and greater. Thus, a decrease in carrageenan content, concomitant with an increase in tissue total P content, was observed, but only at tissue total P levels below 2 mg P·g DW^?1. As these levels were always higher than 2 mg P·g DW^?1 in the winter, spring, and summer experiments, carrageenan content remained constant within each season at 46.2 ± 1.3, 43.1 m 0.7, and 44.5 ± 0.6% DW, respectively. Nitrogen enrichment of plants collected in the fall did not affect carrageenan content, which was stable at 49.3 ± 0.9% DW. When these plants were compared with those of the previous fall experiment (6 μM P·wk^?1 and 53.5 μM N·wk^?1), a slight increase in carrageenan content was noted. Thus, at sufficiently high concentration, N also decreased carrageenan content in C. crispus. Phosphorus nutrition had no significant effect on photosynthesis versus irradiance parameters (P_max, α, R_d, I_c, and I_k), the contents of the photosynthetic pigments chlorophyll-a, phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC), and the ratios PE:APC and PC:APC. In contrast, N nutrition affected both P_maxand the photosynthetic pigment contents. The data indicate that N limitation reduces the number of phycobilisomes but not their size. The greater reduction in phycobiliprotein than chlorophyll-acontent corroborates the natural bleaching phenomenon regularly observed in C. crispus populations during summer when N levels are generally low in seawater. These results suggest that C. crispus in the temperate waters of the Bay of Fundy may experience N limitation, but P limitation is unlikely.     

Properties of auricularia auricula-judae β-D-glucan in dilute solution

A Water-soluble glucan A was isolated from the fruit body of Auricularia auricula-judae. It is composed of a backbone chain of β-(1 → 3)-linked D -glucose residues, two out of three glucose residues being substituted at C-6 positions with a single glucose unit. The weight average molecular weight Mw, number average molecular weight Mn, and intrinsic viscosity [η] of the fractionated samples were studied at 25°C in water and in dimethylsulfoxide (DMSO). The Mark-Houwink equation was established as [η] = 6. 10 × 10^?4 Mw^1.14 for the glucan A having Mw ranging from 9 × 10⁵ to 1.6 × 10⁶ in water. The values of [η] in water are far higher than those in DMSO, but the values of Mn measured in water are the same as those in DMSO. Analysis of Mw and [η] in terms of the known theories for rods and wormlike chains yielded 1030 ± 100 nm^?1, 90 ± 20 nm, 1.3 ± 0.3 nm, and 0.26 ± 0.03 nm for molar mass per unit contour length M_L, persistence length q, diameter d, and contour length h per main-chain glucose residue, respectively. The present data suggest that glucan A dissolves in water as single-stranded helical chains and in DMSO as semiflexible chains. © 1995 John Wiley & Sons, Inc.     

Heterocyst division in two blue-green algae

Bacteriophage 16-6-12 of Rhizobium lupini has a long, non-contractile tail and a head which is hexagonal in outline. The tail is 140 nm in length, 11 nm in diameter, and carries a short terminal fiber. Analysis of the tail structure by optical diffraction indicates that it is of the helical “stacked disc” type. After phenol-extraction from purified particles, the DNA of phage 16-6-12 can circularize in vitro. No significant difference in contour length was observed between the linear (14.34±0.28 μm) and circular (14.44±0.24 μm) forms of molecules. After partial denaturation with alkali an AT-GC-map was constructed, which shows an asymmetric distribution of AT- and GC-rich regions. It is concluded that this phage DNA can circularize due to the presence of cohesive ends and that it is not circularly permuted.     

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.     

Characterization of Virion RNAs of Southern Bean Mosaic Virus by Electron Microscopy

Electron microscopy of denatured RNA of southern bean mosaic virus (SBMV) shows two principal linear components, 0.31 ± 0.08 μm (subgenomic RNAs, MW 0.51 × 10⁶) and 0.80 ± 0.17 μm (genomic RNA, MW 1.3 × 10⁶ 25S). Nondenatured RNA (～ 32S) from heat-inactivated virions measure 1.0 ± 0.20 μm (MW 1.64 × 10⁶) but is poorly-infectious. Upon denaturation the 325 RNA disaggregates into components of lengths typical of the genomic and subgenornic RNAs and infectivity is restored.     

Abnormality of 2-deoxyglucose uptake kinetics in fibroblasts at low concentrations

2-deoxyglucose uptake rates at low sugar concentrations (less than 500 μM) appeared to be lower than those predicted by the Michaelis-Menten model which correctly described higher concentrations. This phenomenon which we will call concentration-dependent transport lag, was also observed for L-glucose uptake which suggest that this phenomenon is carrier-independent. A model involving the perimembrane space is developed which, for L-glucose, gives k₁ = 0.931 ± 0.072 × 10^?6 l. mg protein^?1. minute^?1, k₂ = 2.97 ± 0.19 × 10^?7 l. mg protein^?1. minute^?1 and So = 88,8 ± 4,3 μM; where k₁ is the diffusion constant in the cell membrane, k₂ is the diffusion constant in the perimembrane space and S_o the sugar concentration required in the external medium in order to provide an équivalent sugar concentration in the transport carrier area.     

Forskolin-loaded human serum albumin nanoparticles and its biological importance

Abstract

In this study, forskolin-loaded human serum albumin nanoparticles (FR-HSANPs) were successfully prepared by incorporation and affinity-binding methods. FR-HSANPs were characterized by transmission electron microscope that most of them are circular in shape and size is around 340?nm. The drug loading was more than 88% and further sustained release profiles were observed as it is 77.5% in 24?h time. Additionally, the cytotoxicity results with HepG2 cells indicated that FR-HSANPs showed significantly higher cytotoxicity and lower cell viability as compared to free forskolin (FR). Furthermore, to understand the binding mechanism of human serum albumin (HSA) with forskolin resulted from fluorescence quenching as a static mechanism and the binding constant is 6.26?±?0.1?×?10⁴ M^?1, indicating a strong binding affinity. Further, association and dissociation kinetics of forskolin–HSA was calculated from surface plasmon resonance spectroscopy and the binding constant found to be K_forskolin = 3.4?±?0.24?×?10⁴ M^?1 and also fast dissociation was observed. Further, we used circular dichroism and molecular dynamics simulations to elucidate the possible structural changes including local conformational changes and rigidity of the residues of both HSA and HSA–forskolin complexes.

Communicated by Ramaswamy H. Sarma     

Intracellular Metabolism of β-L-ddAMP-bis(tbutylSATE), a Potent Inhibitor of Hepatitis B Virus Replication

Abstract

β-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC₅₀ = 0.1 μM. Following a 0-to72-hrs exposure of human hepatocytes to a 10 μM [2′,3′^?3H] β-L-ddAMP-bis(tbutylSATE), the pharmacologically active β-L-ddATP was the predominant metabolite attaining a concentration of 268.53 ± 107.97 pmoles/10⁶ cells at 2 hrs. In Hep-G2 cell, β-L-ddATP accounted for 146.8 ± 29.8 pmoles/10⁶ cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of β-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.     

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of ±1.5% of full scale in 65 536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 μm when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 · 10^?4 torr^?1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 · 10^?4 torr^?1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficent of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.