Uptake of dimannoside clusters and oligomannosides by human dendritic cells

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the -amino group of -fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six -D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys₅-Ala-Cys-NH₂). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.     

The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C Virus Helicase NS3h Is Dependent on Both Sugar and Base Moieties

The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3′-to-5′ superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s^− 1 (oligo(dT)), 35 nt s^− 1 (oligo(dU)), and 42 nt s^− 1 (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K_0.5 for NS3h translocation at a saturating ATP concentration are as follows: 3.3 ± 0.4 μM nucleotide (poly(dT)), 27 ± 2 μM nucleotide (poly(dU)), and 36 ± 2 μM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS⁰ are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (∼ 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.     

Glycoconjugates of the human sperm surface: Distribution and alterations that accompany capacitation in vitro

We have studied changes in the binding of fluoresceinated lectins to human sperm during in vitro capacitation. We first determined the surface labeling pattern of viable sperm obtained by the swim-up procedure. Sperm were labeled with 100 μg/ml FITC-conjugated lectin at 4°C for 30 min. We simultaneously used Hoechst stain 33258 as a supravital stain to help differentiate surface from intracellular lectin labeling. Of 14 lectins studied, six (phytohemagglutinin-E, concanavalin A, Ricinus communis agglutinin-I, and the lectins of wheat germ, Lens culinaris, and Pisum sativum) bound to the entire surface of sperm, sometimes with minor local heterogeneity. Three lectins (from peanut, Maclura pomifera, and soybean) usually bound in a punctate manner, with more label on the tail than on the head. Five lectins (Ulex europaeus, Dolichos biflorus, Helix pomatia, and Vicia villosa lectins, and lectin II of Griffonia simplicifolia) bound very poorly or not at all to the sperm surface. Sperm were also inspected for changes in surface lectin binding patterns after 0, 5, and 23 hr of incubation in a capacitating medium. Two lectins showed reproducible changes. The labeling by Maclura pomifera agglutinin decreased by 5 hr in eight of ten experiments, and among sperm labeled with concanavalin A, the incidence of sperm with a highly fluorescent anterior margin of the sperm head increased by about 3.5-fold between 0 and 5 hr. The labeling pattern of the other lectins did not change.     

Influence of Ageing and Abscisic Acid on Potassium Uptake by Potato Tuber Tissues

The potassium uptake by potato tuber discs tissues freshly cut and after 24 h of ageing in the presence or not of abscisic acid was investigated. Uptake kinetics revealed a biphasic dependence on external K⁺ concentrations. At concentration less than 10 mM, uptake was mediated by a saturable component and a linear component became apparent at higher concentrations. At low K⁺ concentrations (lmM), the capacity of K⁺ uptake diminished by 2 times after ageing. Treatment of tissues with ABA increased the rate of K⁺ uptake. In both fresh and aged tissues the uptake was strongly enhanced by fusicoccin and decreased by several metabolic inhibitors and ATPase inhibitors, underlying the active nature of uptake and suggesting the involvement of a plasmalemma H⁺-ATPase in K⁺ transport system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spacer Length Dependence on the Efficiency of Dimeric Anionic Peptides in Gene Transfer by Glycosylated Polylysine/Plasmid Complexes

Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol. It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-Ala-Ala residues, but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the cells to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.     

Intercalating Nucleic Acids: The Influence of Linker Length and Intercalator Type on Their Duplex Stabilities

Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.     

Influence of Diethylaminoethyl-Dextran on Uptake and Degradation of Polyoma Virus Deoxyribonucleic Acid by Mouse Embryo Cells

The uptake of (32)P-labeled polyoma virus deoxyribonucleic acid (DNA) (I and II + III) by mouse embryo cells was increased from two- to fivefold in the presence of 500 mug of diethylaminoethyl-dextran (DEAE-D) per ml. This concentration of DEAE-D gives maximal enhancement of infectivity; however, the increase is many thousand-fold. As the DEAE-D concentration was increased from 0 mug/ml, uptake and infectivity increased to flat maxima and then decreased in a similar manner, except that at low DEAE-D concentrations uptake was relatively much greater than infectivity. Several other polycations also increased DNA uptake but did not enhance infectivity, and uptake of viral DNA was unaffected by the presence of mouse DNA, although infectivity was reduced. Thus, increased uptake is not the sole basis for the enhancement of infectivity produced by DEAE-D. The possibilities that DNA complexed with DEAE-D penetrates more rapidly or is stabilized against degradation do not completely account for enhancement since complexes formed in mixtures of DNA and DEAE-D, which sedimented heterogeneously from 40 to 60S, were infectious only for monolayers treated with DEAE-D. A more likely factor in enhancement is inhibition of the cellular nuclease activity detected, since virus DNA exposed to cells was much more degraded in the absence than in the presence of DEAE-D. The nuclease activity produced single-strand breaks in double-stranded DNA. Treatment of monolayers with deoxyribonuclease after adsorption of DNA in the presence of DEAE-D reduced cell-associated radioactivity by about 70%, although the number of plaques formed was not affected. In the absence of DEAE-D, 90 to 100% was removed by deoxyribonuclease. Thus, in both cases most of the DNA was adsorbed extracellularly. The greater deoxyribonuclease-resistant fraction in the presence of DEAE-D would be consistent with another possibility: that enhancement results from an increase in DNA penetration rate due to some action of DEAE-D on the cell.     

桃果实不同发育时期细胞内糖酸分布对甜酸风味的影响

为了解桃果实发育过程中细胞内糖酸的分布、变化规律对果实甜酸风味的影响,采用区室分析方法研究了‘白凤’桃(Prunus persica‘Hakuho’)果实不同发育时期细胞内糖酸组分、含量及其分布对甜酸风味的影响。结果表明,成熟果实中(花后100 d)可溶性糖(蔗糖、葡萄糖、果糖和山梨醇)在液泡、细胞质和细胞间隙中的含量分别为27.3、11.6、9.0 mg/g,有机酸(苹果酸、柠檬酸、奎宁酸和莽草酸)含量为2.09、0.94、0.35 mg/g;未成熟果实中(花后60 d)可溶性糖在液泡、细胞质和细胞间隙中的含量分别为0.97、2.2、2.3 mg/g,有机酸含量为0.25、0.44、0.82 mg/g。‘白凤’桃果肉细胞内不同的糖酸分布对成熟果实的甜酸风味具有显著影响,而对未成熟果实影响较小。成熟果实中糖酸在液泡、细胞质和细胞间隙三者之间的分布差异可能是导致果实甜度变化的主要原因。     

Hydrolysis and Esterification with Lipase from Candida Cylindracea. Influence of the Reaction Conditions and Acid Moiety on the Enantiomeric Excess

The enantiomeric ratio for hydrolysis and synthesis of 1-phenyl ethanol esters of straight chain aliphatic carboxylic acids catalyzed by Candida cylindracea lipase was determined. A distinct maximum in enantiomeric ratio was observed for valeric and caproic acid in the hydrolytic direction. No significant maximum could be determined in the esterification reaction. Even though the enzyme provided larger enantiomeric ratios in the synthetic direction the enantiomeric excess of the alcohol was not higher. The enantiomeric excess was depressed by racemization reactions in the esterification as the reaction approached thermodynamic equilibrium at an insufficient conversion. While choosing the optimal chain length of the acyl donor is important in hydrolytic reactions it seems to be of greater value to raise the equilibrium conversion in the esterification reactions.     

蛋白核酸合成抑制剂和蛋白磷酸化对基因表达的调节

环己酰亚胺和放线菌素D明显降低小麦幼苗中BADH基因的表达,表明BADH基因表达在转录和转译水平上受到调控。氯霉素则有增加表达的效应。线粒体可能形成阻遏蛋白参与调节。H7和甘露糖降低BADH基因表达,相应地冈田酸(Okadaic acid)明显增加表达,说明蛋白磷酸化积极参与小麦幼苗中BADH基因表达的调节。     

Influence of Polymers on the Physical and Chemical Stability of Spray-dried Amorphous Solid Dispersion: Dipyridamole Degradation Induced by Enteric Polymers

Amorphous solid dispersions (ASDs) are inherently unstable because of high internal energy. Evaluating physical and chemical stability during the process and storage is essential. Numerous researches have demonstrated how polymers influence the drug precipitation and physical stability of ASDs, while the influence of polymers on the chemical stability of ASDs is often overlooked. Therefore, this study aimed to investigate the effect of polymers on the physical and chemical stability of spray-dried ASDs using dipyridamole (DP) as a model drug. Proper polymers were selected by assessing their abilities to inhibit drug recrystallization in supersaturated solutions. HPMC E5, Soluplus®, HPMCP-55, and HPMCAS-LP were shown to be effective stabilizers. The optimized formulations were further stored at a high temperature (60 °C) and high humidity (40 °C, 75% RH) for 2 months, and their physical and chemical stability was evaluated using polarizing optical microscopy, FTIR, HPLC, and mass spectrometry (MS). In general, crystallization was observed in all samples, which indicated the physical instability under stressed storage conditions. Also, it was noted that the polymers in ASDs rather than physical mixtures, induced a dramatic drug degradation after being exposed to a high temperature (HPMCP-55 >?80% and HPMCAS-LP >?50%) and high humidity (HPMCP-55 >?40% and HPMCAS-LP >?10%). The MS analysis further confirmed the degradation products, which might be generated from the reaction between dipyridamole and phthalic anhydride decomposed from HPMCP-55 and HPMCAS-LP. Overall, the exposure of ASDs to stressed conditions resulted in recrystallization and even the chemical degradation induced by polymers.     

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.     

Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension-cultured Acer pseudoplatanus Cells: II. Influence of 2,4-D Metabolism and Intracellular pH on the Control of Cell Division by Intracellular 2,4-D Concentration

The utilization of 2,4-dichlorophenoxyacetic acid (2,4-D) molecules by Acer pseudoplatanus cells is governed mainly by a glucosylation process. Evidence that 2,4-D glucoside molecules are biologically inactive is presented. 2,3,5-Triiodobenzoic acid (TIBA), by inhibiting 2,4-D glucosylation, has a sparing effect on 2,4-D molecules; thus TIBA treatments increase growth yield (expressed as the ratio of the maximum number of cells produced to the initial concentration of 2,4-D in the culture medium).     

Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

Sucrose and Hexose Release by Excised Stem Segments of Vicia faba L.: The Sucrose-Specific Stimulating Influence of Cuscuta on Sugar Release and the Activity of Acid Invertase

By washing out solutes in 0.5 mM CaSO⁴at 25 °C during aperiod of 5–6 h, the release of sugars by excised stemsegments of Vicia faba L. was measured. The stem parasite Cuscutaeuropaea strongly stimulated the release of sucrose into theefflux medium; this effect was most marked during the last hoursof each experiment but this stimulating effect of the parasitecould not be detected for glucose and fructose. The fact thatparasitized stem segments released higher than normal hexoseamounts during the last hours of several experiments, couldbe explained as the result of extracellular hydrolysis of sucroseby free space invertase. A high free space acid invertase activitywas present in young stem segments of Vicia faba and in tissuesof Cuscuta. The stimulating influence of Cuscuta on sugar releaseby cells of stem segments appears sucrose-specific, supportingthe idea that the stimulating influence of Cuscuta on sugarrelease is restricted to the sieve-tube system. When metabolic inhibitors were added to the washing solutionor when segments were incubated at low temperature, no cleareffect of the parasite could be observed and for all segments(parasitized and non-parasitized) a strongly enhanced releaseof sucrose into the efflux medium was found during the lasthours of an experiment. These data support the idea that anintensive resorption of sucrose occurs within stem segments,after its release into the apoplast. Key words: Cuscuta europaea, Parasitic relationship, Phloem unloading     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Influence of extracellular calcium on cell permeabilization and growth regulation by the lymphokine leukoregulin

Permeablization of human K562 leukemia cells was measured in the presence and absence of extracellular ionic calcium to examine the relationship of ionic calcium to increased membrane permeability and the inhibition of cell proliferation by this lymphokine. In the absence of extracellular calcium, the ability of leukoregulin to permeabilize the cell membrane is diminished but is fully restored by addition of 1 mM extracellular Ca++ as shown flow cytometrically by loss of intracellular fluorescein. Membrane permeability is also increased by calcium ionophore A23187 but permeablization is completely blocked in calcium-free medium despite the intramembrane presence of the calcium ionophore. Membrane permeablization by the lectin phytohemagglutinin, in contrast, is independent of extracellular calcium. A similar divergence in cell proliferation activity of the three modulators of calcium flux and membrane permeability occurs in the absence of extracellular calcium. Leukoregulin inhibition of cell proliferation is abolished, inhibition by calcium ionophore A23817 is greatly reduced, and inhibition by phytohemagglutinin is unchanged. Leukoregulin permeabilized K562 cells isolated by fluorescence activated cell sorting resume proliferation after 72 h. In contrast cells permeablized by calcium ionophore A23187 or phytohemagglutinin fail to resume proliferation by 7 days. The membrane permeablizing action of leukoregulin is, therefore, partially dependent upon extracellular calcium. It is also effected through a mechanism other than calcium ionophore transport or lectin type transmembrane signaling, and is accompanied by a reversible inhibition of cell proliferation.