Preparation and Characterization of Copolymer Micelles for the Solubilization and <Emphasis Type="Italic">In Vitro</Emphasis> Release of Luteolin and Luteoloside

Luteolin (LUT) and luteoloside (LUS) belong to flavonoids with high anticancer potential and were loaded into biodegradable diblock copolymer micelles of methoxy polyethylene glycol-polycaprolactone (mPEG_5K-PCL_10K), methoxy polyethylene glycol-polylactide-co-glycolide (mPEG_5K-PLGA_10K), and methoxy polyethylene glycol-polylactide (mPEG_5K-PDLLA_10K) by a self-assembly method, creating water-soluble LUT and LUS copolymer micelles, respectively. The solubilization formulations of the copolymer micelles were optimized with response surface methodology (RSM). The obtained drug micelles are torispherical under transmission electron microscope (TEM) with an average diameter of about 70 nm. The mPEG_5K-PLGA_10K exhibited higher loading capacity for LUS which was 4.33%, and LUT- (or LUS)-loaded mPEG_5K-PCL_10K exhibited a better stability and encapsulation efficiency which was 65.1 and 55.8%, respectively. The in vitro drug release study showed above 47% of LUT was released from micelles at pH 7.4 PBS; however, no more than 35% of LUT was released at pH 6.4 PBS within 24 h. Meanwhile, no more than 30% of LUS was released from micelles whether at pH 6.4 or 7.4 PBS solution within 24 h.     

Folate-functionalized unimolecular micelles based on a degradable amphiphilic dendrimer-like star polymer for cancer cell-targeted drug delivery

A folate-functionalized degradable amphiphilic dendrimer-like star polymer (FA-DLSP) with a well-defined poly(L-lactide) (PLLA) star polymer core and six hydrophilic polyester dendrons based on 2,2-bis(hydroxymethyl) propionic acid was successfully synthesized to be used as a nanoscale carrier for cancer cell-targeted drug delivery. This FA-DLSP hybrid formed unimolecular micelles in the aqueous solution with a mean particle size of ca. 15 nm as determined by dynamic light scattering and transmission electron microscopy. To study the feasibility of FA-DLSP micelles as a potential nanocarrier for targeted drug delivery, we encapsulated a hydrophobic anticancer drug, doxorubicin (DOX), in the hydrophobic core, and the loading content was determined by UV-vis analysis to be 4 wt %. The DOX-loaded FA-DLSP micelles demonstrated a sustained release of DOX due to the hydrophobic interaction between the polymer core and the drug molecules. The hydrolytic degradation in vitro was monitored by weight loss and proton nuclear magnetic resonance spectroscopy to gain insight into the degradation mechanism of the FA-DLSP micelles. It was found that the degradation was pH-dependent and started from the hydrophilic shell gradually to the hydrophobic core. Flow cytometry and confocal microscope studies revealed that the cellular binding of the FA-DLSP hybrid against human KB cells with overexpressed folate-receptors was about twice that of the neat DLSP (without FA). The in vitro cellular cytotoxicity indicated that the FA-DLSP micelles (without DOX) had good biocompatibility with KB cells, whereas DOX-loaded micelles exhibited a similar degree of cytotoxicity against KB cells as that of free DOX. These results clearly showed that the FA-DLSP unimolecular micelles could be a promising nanosize anticancer drug carrier with excellent targeting property.     

PSMA Ligand Conjugated PCL-PEG Polymeric Micelles Targeted to Prostate Cancer Cells

In this content, a small molecular ligand of prostate specific membrane antigen (SMLP) conjugated poly (caprolactone) (PCL)-b-poly (ethylene glycol) (PEG) copolymers with different block lengths were synthesized to construct a satisfactory drug delivery system. Four different docetaxel-loaded polymeric micelles (DTX-PMs) were prepared by dialysis with particle sizes less than 60 nm as characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Optimization of the prepared micelles was conducted based on short-term stability and drug-loading content. The results showed that optimized systems were able to remain stable over 7 days. Compared with Taxotere, DTX-PMs with the same ratio of hydrophilic/hydrophobic chain length displayed similar sustained release behaviors. The cytotoxicity of the optimized targeted DTX-PCL_12K-PEG_5K-SMLP micelles (DTX-PMs2) and non-targeted DTX-PCL_12K-mPEG_5K micelles (DTX-PMs1) were evaluated by MTT assays using prostate specific membrane antigen (PSMA) positive prostate adenocarcinoma cells (LNCaP). The results showed that the targeted micelles had a much lower IC50 than their non-targeted counterparts (48 h: 0.87±0.27 vs 13.48±1.03 µg/ml; 72 h: 0.02±0.008 vs 1.35±0.54 µg/ml). In vitro cellular uptake of PMs2 showed 5-fold higher fluorescence intensity than that of PMs1 after 4 h incubation. According to these results, the novel nano-sized drug delivery system based on DTX-PCL-PEG-SMLP offers great promise for the treatment of prostatic cancer.     

Effects of Ellman’s reagent and other thiol compounds on ion transport and ATPase activity in anaerobically grown Escherichia coli cells

It was found that modification of thiol (SH-) groups of membrane proteins by Ellman’s reagent (5,5′-dithiol-bis-(2-nitrobenzoic) acid) results in inhibition of proton efflux and K⁺ influx in anaerobically grown (pH 7.5) wild-type strains of Escherichia coli and causes disturbances in K⁺-dependent, N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity and molecular hydrogen production. No such effects were observed after substitution of the cysteine residue in the b-subunit of F₀ of proton F₀F₁-ATPase for alanine. Moreover, the redox potential (RP) decreased as a result of H₂ release during glucose fermentation and formate utilization was partly restored in the presence of Ellman’s reagent. Similar changes were established when another specific SH-reagent, succinimidyl-6(β-maleimidopropionamido)hexanoate, was used. Another thiol reagent, N-ethylmaleimide, did not exert such effects despite its inhibitory action on ion transport and ATPase activity. The data obtained provide conclusive evidence in favor of essential role of thiol groups and the cysteine residue in the b-subunit of F₀ of F₀F₁-ATPase in proton-potassium exchange and H₂ production in E. coli cells. The results also point to a possible involvement of SH-groups in the TrkA system of K⁺ uptake and an involvement of hydrogenases 3 or 4 in the interactions of these integral proteins with each other.     

Spin Trapping of Ibuprofen Radicals: Evidence That Ibuprofen is a Hydroxyl Radical Scavenger

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2–(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vim. Ibuprofen (IBU; 0.1–50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5–2.0mM). and either 5.5-dimethyl-l-pyrroline-N-oxide (DMPO; 40mM) or a-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (l mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (a_N = 16.1G, a^H_β = 24.0G) and PBN (a_N = 15.7G. a^H_β = 4.4G and a_N = 17.0G, a^H_β = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (l mM), hydrogen peroxide (lmM), PBN (48mM), and increasing IBU concentrations. Therefore. we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hydroxyl radicals would be generated, less IBU radicals formed and the signal height of IBU radicals trapped by PBN would have decreased. However, these data do not fully exclude the possiblity that IBU may, to some extent. also chelate iron. Scavenging of hydroxyl radicals may be one of the mechanisms responsible for the beneficial action of IBU during the management of several rheumatic diseases. However, the IBU radicals produced when IBU scavenges hydroxyl radicals are reactive. and may be associated with the reported toxicity of this therapeutic agent.     

Zero extracellular K+ and prostaglandin E release in the guinea-pig taenia coli

Prostaglandin E release rates from isolated strips of guinea-pig taenia coli increased during exposure to zero K⁺ bathing fluid, from control values of $\text{0.78 ± 0.11}\text{ng/g}$ per min to levels as high as 29.2 ng/per min. Release rates increased for 40–50 min and then remained constant or fell despite progressive increases in intracellular sodium [Na_i⁺] or fall in intracellular potassium [K_i⁺]. Readmittance of K⁺ to the bathing solution resulted in rapid reversal of elevated prostaglandin E release rates. [Na⁺_i] and [K⁺_i] were markedly more abnormal in strips exposed to zero K⁺ for 70–201 min compared to 30-min exposures. Upon the readdition of K⁺ after long zero K⁺ exposure, the rate of prostaglandin E release fell long before [Na⁺_i] and [K⁺_i] returned to control levels. After K⁺ was readded to the bathing solution, the ion concentration of tissues exposed to zero K⁺ for 30 min returned to normal much more quickly than did those of tissues exposed for the longer time periods, yet the exponential rate constants for fall of prostaglandin E release rate after K⁺ was added were not significantly different after short or long zero K⁺ exposure. Thus there was a dissociation between the return of [Na⁺_i] and [K⁺_i] and the fall of prostaglandin E release rate to control levels. Ouabain augmented prostaglandin E release under conditions where [K⁺_i] could not fall. Addition of known neurotransmitters present in this tissue to the bathing fluid did not augment prostaglandin E release. Guinea-pig taenia coli strips that had been incubated with [³H]arachidonic acid, constantly released [³H]arachidonic acid and [³H]prostaglandin E and a prostaglandin which cochromatographed with prostaglandin E but could not be converted to prostaglandin B by alkali and was shown to be 6-ketoprostaglandin F_1α. Release of [³H]arachidonic acid and [³H]prostaglandin E plus 6-[³H]ketoprostaglandin F_1α was increased when strips were exposed to zero K⁺. Data obtained in this study suggest the augmented prostaglandin E release seen during zero K⁺ or ouabain is related to increased availability of unbound arachidonic acid at the site of cyclooxygenase in the cell. Augmented prostaglandin E release is apparently not related to alterations in intracellular electrolyte concentrations or release of known neurotransmitters.     

Unusual titration of the membrane-bound artificial hemagglutinin fusion peptide

E5 is a 20-residue-long analog of the fusion peptide from influenza hemagglutinin (GLFEAIAEFIEGGWEGLIEG). It has been suggested that two of its five glutamates, Glu11and Glu15, are critical in its pH-dependent membrane perturbation. To reveal their specific involvement, a pair of analogs with substitution of either Glu11 or Glu15 for Ala were synthesized. By analysis of the pH-dependence of the chemical shifts of protons of these peptides bound to dodecylphosphocholine micelles we found: (1) the peptides adopt an amphiphilic alpha-helical structure within residues 2?C18, similar to the parent peptide; (2) the helix is significantly more disordered at neutral pH than at acidic pH for E5 peptide only; and (3) in E5 and mutant peptides the Glu11 and 15 residues have similar pK _a values, higher than those of the other glutamates. This excludes their mutual interaction in E5, being a source of the elevated pK _a values. We attribute this phenomenon to the presence of minor states caused by deepening of the Glu11 and 15 side-chains in the hydrophobic environment of the membrane. As the mid-pH of membrane-perturbation activity of E5 matches the pK _a value of these glutamates, we conclude their presence contributes to the plasticity of the peptide and determines the pH-dependence of membrane perturbation caused by E5.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

The amino- and carboxyl-terminal sequence of bovine rhodopsin

The amino terminus of bovine rhodopsin is blocked and has the sequence x-Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys-Thr-Gly-Val-Val-Arg, where CHO represents sites of carbohydrate attachment. The carboxyl-terminal sequence of rhodopsin is Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala. Upon short-term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (～ 35,000 daltons) is converted to a membrane-bound fragment O′ (～ 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long-term digestion of ROS with thermolysin, opsin and O′ are replaced by the membrane-bound fragments F₁ (～25,000 daltons), and F₂ (～9,500 daltons). When ³²P-ROS are digested, F₂ carries the ³²P. Both O′ and F₁ contain the amino-terminal glycopeptide.     

Targeted Delivery of Doxorubicin-Loaded Poly (ε-caprolactone)-b-Poly (N-vinylpyrrolidone) Micelles Enhances Antitumor Effect in Lymphoma

Background

The present study was motivated by the need to design a safe nano-carrier for the delivery of doxorubicin which could be tolerant to normal cells. PCL₆₃-b-PNVP₉₀ was loaded with doxorubicin (6 mg/ml), and with 49.8% drug loading efficiency; it offers a unique platform providing selective immune responses against lymphoma.

Methods

In this study, we have used micelles of amphiphilic PCL₆₃-b-PNVP₉₀ block copolymer as nano-carrier for controlled release of doxorubicin (DOX). DOX is physically entrapped and stabilized in the hydrophobic cores of the micelles and biological roles of these micelles were evaluated in lymphoma.

Results

DOX loaded PCL₆₃-b-PNVP₉₀ block copolymer micelles (DOX-PCL₆₃-b-PNVP₉₀) shows enhanced growth inhibition and cytotoxicity against human (K-562, JE6.1 and Raji) and mice lymphoma cells (Dalton''s lymphoma, DL). DOX-PCL₆₃-b-PNVP₉₀ demonstrates higher levels of tumoricidal effect against DOX-resistant tumor cells compared to free DOX. DOX-PCL₆₃-b-PNVP₉₀ demonstrated effective drug loading and a pH-responsive drug release character besides exhibiting sustained drug release performance in in-vitro and intracellular drug release experiments.

Conclusion

Unlike free DOX, DOX-PCL₆₃-b-PNVP₉₀ does not show cytotoxicity against normal cells. DOX-PCL₆₃-b-PNVP₉₀ prolonged the survival of tumor (DL) bearing mice by enhancing the apoptosis of the tumor cells in targeted organs like liver and spleen.     

Spectroscopic investigation of europium(III) nitrato complexes in anhydrous and aqueous acetonitrile

Electronic absorption and emission spectra, along with lifetime measurements and vibrational spectra, are used to investigate the interaction between nitrate and trivalent europium ions in dilute solutions in anhydrous and aqueous acetonitrile. Upon addition of increasing quantities of nitrate, the complexes [Eu(NO₃)_n]^(3?n)+, with n = 1–5, form quantitatively in anhydrous acetonitrile. In solution, the pentanitrato species is not further solvated and its spectroscopic properties are similar to those of solid samples, indicating a similar structure with five bidentate nitrates bonded to the 10-coordinate Eu(III) ion. The lifetimes of the ⁵D₀ level are 1.35(5) and 1.25(5) ms for Eu(NO₃)₃ and (Me₄N)₂Eu(NO₃)₅ 0.05 M in CH₃CN. The quantum yield of Eu(NO₃)₃ in CH₃CN is 27.4%.The addition of small quantities of water to Eu(NO₃)₃ solutions does not result in the dissociation of the nitrate ions, provided R_w = [H₂O]_t/[Eu³⁺]_t is smaller than 8; the apparent equilibrium rations for [Eu(NO₃)₃(H₂O)_n] are K₃ = 40 ± 15 M^?1 and K₄ = 9 ± 3 M^?1; K₁ and K₂ are too large to be determined. The formation of nitrato complexes is studied in mixtures containing increasing amounts of water and nitrate. Deconvolution of the different components of the ⁵D₀ → ⁷F₀ transition allows a semi-quantitative estimate of the relative concentration of the nitrato complexes. The total number of coordinated nitrate ions per europium ion can be determined on the basis of fluorescence lifetime measurements. The apparent equilibrium ratios for the formation of the mono- and dinitrato species amount to K₁ = 23 ± 3, 15 ± 5 and 5 ± 1 for R_w = 44, 94 and 304, respectively, and to K₂ = 17 ± 8 for R_w = 44 and 94.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[α-P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[α-³²P]ADP in the dark with a K_d value of 8 μM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[α-³²P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[α-³²P]ADP, both the ADP/ATP carrier and the β subunit of the membrane-bound F₁-ATPase are covalently labeled. The binding specificity of 2-azido-[α-³²P]ADP for the β subunit of F₁-ATPase is ascertained by prevention of photolabeling of isolated F₁ by preincubation with an excess of ADP.     

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 µg/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 ^oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.     

Kinetic analysis for the isomerization of glucose,fructose, and mannose in subcritical aqueous ethanol

Fructose, glucose, and mannose were treated with subcritical aqueous ethanol for ethanol concentrations ranging from 0 to 80% (v/v) at 180–200 °C. The aldose–ketose isomerization was more favorable than ketose–aldose isomerization and glucose–mannose epimerization. The isomerization of the monosaccharides was promoted by the addition of ethanol. In particular, mannose was isomerized most easily to fructose in subcritical aqueous ethanol. The apparent equilibrium constants for the isomerizations of mannose to fructose, K_eq,M→F, and glucose to fructose, K_eq,G→F, were independent of ethanol concentration and increased with increasing temperature. Moreover, the K_eq,M→F value was much larger than the K_eq,G→F value. The enthalpies for the isomerization of mannose to fructose, ΔH_M→F, and glucose to fructose, ΔH_G→F, were estimated to be 18 and 24 kJ/mol, respectively, according to van’t Hoff equation. Subcritical aqueous ethanol can be used to produce fructose from glucose and mannose efficiently.     

Relationships between xylem anatomy,root hydraulic conductivity,leaf/root ratio and transpiration in citrus trees on different rootstocks

The aim of the study was to determine the extent in which leaf and whole plant transpiration (Tp) were influenced by root hydraulic conductance (K_r), leaf to root ratio and leaf mass. Also, the relationships between the anatomic characteristics of roots and K_r were investigated. To this end, 9‐month‐old seedlings of the citrus rootstocks Cleopatra mandarin (CM), Poncirus trifoliata (PT), and their hybrids Forner‐Alcaide no 5 (FA‐5) and Forner‐Alcaide no 13 (FA‐13) and 15‐month‐old trees of Valencia orange budded on these four rootstocks were tested. The hybrid FA‐13 and PT had higher values of K_r and leaf transpiration rates (E) than FA‐5 and CM. There was a positive curvilinear correlation between E and K_r. Furthermore, E levels in the different types of plants decreased with increased leaf/root (L/R) ratios. Pruning of the roots and defoliation confirmed that transpiration rates were strongly influenced by the L/R ratio. However, variations in E because of differences in L/R ratios were less pronounced in trees budded on FA‐13 and PT than on the other two rootstocks. In addition, there was a positive correlation between Tp and leaf biomass, although differences between rootstocks may be attributed to differences in K_r. The average lumen diameter of xylem vessels was greater in rootstocks with high K_r. Size of epidermal and hypodermal cells of fibrous roots may also restrict K_r.     

Resolution and Reconstitution of H+-ATPase Complex from Beef Heart Mitochondria

Mitochondrial H⁺-ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a “membrane” (NaBr-F₀) and a “soluble” fraction by treatment with 3.5 M sodium bromide. The NaBr-F₀ fraction is completely devoid of p, 8, and e subunits of the F, ATPase and largely devoid of α and γ subunits of F₁, where F₀ is used to denote the membrane fraction and F₁, coupling factor 1. This is confirmed by complete loss of ATPase and P₁-ATP exchange activities. The addition of F₁ (400 μg · mg^?1 F₀) results in complete restoration of oligomycin sensitivity without any reduction in the F₁-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F₁-F₀ complex consequent to sodium bromide extraction. Restoration of Pf-ATP exchange and H⁺-pumping activities require coupling factor B in addition to FpATPase. The oligomycin-sensitive ATPase and 32P₁ATP exchange activities in reconstituted Fr F₀ have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F₁-F₀ complex. The data suggest that the altered properties of NaBr-F₀ observed in other laboratories are probably inherent to their F₁F₀ preparations rather than to sodium bromide treatment itself.

The H⁺-ATPase (F₁-F₀) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35–37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F₁, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler-and oligomycin-insensitive. The membrane-bound hydrophobic component F₀, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and P₁-ATP exchange activities on binding to F¹-ATPase (33). The purest preparations of bovine heart mitochondrial F₀ show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial Fo is not known.

The F₀ preparations from bovine heart reported so far have been derived from H⁺-ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37). The ATPase and P₁-ATP exchange activity of the preparations so obtained are low, dependent upon additional phospholipids and coupling factors; they show altered sensitivity to energy transfer inhibitors as compared to submitochondrial particles from the heavy layer of the mitochondria or ETPh (1. 2, 12, 14, 29, 33). Recently, lysolecithin has been successfully employed to extract highly active H⁺-ATPase from beef (17, 19, 28) and pig (24) heart mitochondria. The beef heart H⁺-ATPase preparation has the same ratio of ATPase to PrATP exchange activity and apparently the same sensitivity to energy transfer inhibitors as submitochondrial particles (17). The present communication describes resolution of this F₁-F₀ preparation using sodium bromide (NaBr) and reconstitution of ATPase and Pr ATP exchange activities. The NaBr-F₀ prepared from this preparation shows no dependence on lipids, and the same or increased sensitivity to energy transfer inhibitors when reconstituted with F₁-ATPase. Furthermore, F₁ ATPase activity does not decrease on binding of F₁ to NaBr-F₀, even though the reconstituted ATPase activity is 99% sensitive to oligomy-cin and dicyclohexylcarbodiimide. These properties are in contrast to the properties of F₀ reported by other workers (12, 14).     

Formate Hydrogenlyase Is Needed for Proton-Potassium Exchange Through the F0F1-ATPase and the TrkA System in Anaerobically Grown and Glycolysing Escherichia coli

Anaerobically grown and glycolysing Escherichia coli produced H₂ and carried out H⁺-K⁺-exchange in two steps, the first of which had the fixed stoichiometry for DCCD-sensitive fluxes (2H⁺/K⁺), and the second one had a variable stoichiometry for DCCD-sensitive fluxes. H₂ production and the 2H⁺/K⁺-exchange were lost in the ΔfdhF or ΔhycA-H mutant. In the ΔfdhF mutant, H⁺-K⁺-exchange with K _m for K⁺-uptake of 2.3 mM and less K⁺-gradient between the cytoplasm and the medium were observed. H₂ production and H⁺-K⁺-exchange with a high K _m for K⁺-uptake were carried out in the uncD mutant; however, both H₂ production and H⁺-K⁺-exchange were lost in the Δunc or uncE mutant. H₂ production was observed in the trkA trkD kdpA mutant. It was displayed in protoplasts with increased membrane permeability when donor or acceptor of reducing equivalents—formate with DTT or NADH respectively—was added. The F₀F₁ and the TrkA(H) or the F₀ and the TrkA(G) had been assumed to form the united supercomplexes, functioning as a H⁺-K⁺-pump or antiporter respectively (for review see Bioelectrochem Bioenerg 33:1, 1994). Results allow the proposal that H₂ production by FHL has a relationship with the H⁺-K⁺-exchange through a H⁺-K⁺-pump and via an H⁺-K⁺-antiporter. Formate and NADH can serve as a donor and an acceptor of reducing equivalent respectively, for operation of such supercomplexes. Received: 12 December 1996 / Accepted: 19 March 1997     

Effects of fertilization on the growth,photosynthesis, and biomass accumulation in juvenile plants of three coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.) cultivars

We carried out a field experiment in order to study effects of fertilization in juvenile plants of three coffee (Coffea arabica) cultivars in Yunnan, SW China. Fertilization treatments included a control without fertilizer (CK), combinations of three NPK fertilization rates [high fertilization (F_H), medium fertilization (F_M), and low fertilization (F_L) with 135, 90, and 45 g per plant per year, respectively], and at two N:P₂O₅:K₂O ratios (R₁, 1:0.5:0.8; R₂, 1:0.8:0.5). The growth in juvenile plants was not altered by fertilization, with two clear growth peaks being observed in both the height and stem growth rates (RGRs) throughout a year. Both F_M and F_H resulted in significantly higher RGRs in both height and stem diameter compared to F_L and CK in all three cultivars. At the same fertilization rate, the leaf area, branch number, longest branch length, internode number, and biomass of R₂ were higher than those of R₁, and P significantly affected the root biomass and root to shoot ratio. Compared to the F_L treatment, both F_M and F_H treatments resulted in higher net photosynthetic rates and stomatal conductance across seasons, and in higher intrinsic water-use efficiency during the dry season and at the middle of the wet season. Photosynthetic nitrogen-use efficiency at R₂ was higher than that at R₁, but no significant differences were observed between the different fertilization rates. Among the three coffee cultivars, Caturra exhibited the highest height, stem diameter, longest branch length, and internode number. Our results indicated that the optimal N:P₂O₅:K₂O ratio was 1:0.8:0.5 for the juvenile growth of coffee plants. Both F_M and F_H could help optimize the growth and photosynthetic rate of coffee plants, but F_M is suitable for the ecological friendly agriculture and economic sustainability at coffee plantations.     

Development of Chitosan-Based pH-Sensitive Polymeric Micelles Containing Curcumin for Colon-Targeted Drug Delivery

pH-sensitive N-naphthyl-N,O-succinyl chitosan (NSCS) and N-octyl-N,O-succinyl chitosan (OSCS) polymeric micelles carriers have been developed to incorporate curcumin (CUR) for colon-targeted drug delivery. The physical entrapment methods (dialysis, co­solvent evaporation, dropping, and O/W emulsion) were applied. The CUR-loaded micelles prepared by the dialysis method presented the highest loading capacity. Increasing initial amount of CUR from 5 to 40 wt% to polymer resulted in the increase in loading capacity of the polymeric micelles. Among the hydrophobic cores, there were no significant differences in the loading capacity of CUR-loaded micelles. The particle sizes of all CUR-loaded micelles were in the range of 120–338 nm. The morphology of the micelles changed after being contacted with medium with different pH values, confirming the pH-responsive properties of the micelles. The release characteristics of curcumin from all CUR-loaded micelles were pH-dependent. The percent cumulative release of curcumin from all CUR-loaded micelles in simulated gastric fluid (SGF) was limited to about 20%. However, the release amount was significantly increased after contacted with simulated intestinal fluid (SIF) (50–55%) and simulated colonic fluid (SCF) (60–70%). The released amount in SIF and SCF was significantly greater than the release of CUR from CUR powder. CUR-loaded NSCS exhibited the highest anti-cancer activity against HT-29 colorectal cancer cells. The stability studies indicated that all CUR-loaded micelles were stable for at least 90 days. Therefore, the colon targeted, pH-sensitive NSCS micelles may have potential to be a prospective candidate for curcumin delivery to the colon.     

Mechanistic Insights of Zn2+ Storage in Sodium Vanadates

Rechargeable aqueous zinc‐ion batteries (ZIBs) with high safety and low‐cost are highly desirable for grid‐scale energy storage, yet the energy storage mechanisms in the current cathode materials are still complicated and unclear. Hence, several sodium vanadates with NaV₃O₈‐type layered structure (e.g., Na₅V₁₂O₃₂ and HNaV₆O₁₆·4H₂O) and β‐Na_0.33V₂O₅‐type tunneled structure (e.g., Na_0.76V₆O₁₅) are constructed and the storage/release behaviors of Zn²⁺ ions are deeply investigated in these two typical structures. It should be mentioned that the 2D layered Na₅V₁₂O₃₂ and HNaV₆O₁₆·4H₂O with more effective path for Zn²⁺ diffusion exhibit higher ion diffusion coefficients than that of tunneled Na_0.76V₆O₁₅. As a result, Na₅V₁₂O₃₂ delivers higher capacity than that of Na_0.76V₆O₁₅, and a long‐term cyclic performance up to 2000 cycles at 4.0 A g^?1 in spite of its capacity fading. This work provides a new perspective of Zn²⁺ storage mechanism in aqueous ZIB systems.