Electrochemically induced disintegration of layer-by-layer-assembled thin films composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer assembly composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared on the surface of a platinum (Pt) film-coated quartz resonator, and an electrochemically induced disintegration of the avidin-ib-PEI assembly was studied using a quartz crystal microbalance. The resonance frequency of a five-bilayer (avidin-ib-PEI)5 film-coated quartz resonator was increased upon application of an electric potential to the Pt layer of the quartz resonator, suggesting that the mass on the quartz resonator was decreased as a result of disintegration of the (avidin-ib-PEI)5 film, due to a pH change in the vicinity of the surface of the Pt-coated quartz resonator. It may be that the (avidin-ib-PEI)5 film assembly was decomposed by acidification of the local pH on the surface of the Pt layer, which in turn was induced through electrolysis of water on Pt, because ib-PEI forms complexes with avidin only in basic media. In pH 9 solution, the (avidin-ib-PEI)5 film was decomposed under the influence of an applied potential of 0.6-1.0 V versus Ag/AgCl. The (avidin-ib-PEI)5 film was decomposed almost completely within a minute in a low concentration buffer (1 mM, pH 9), while the decomposition was slower in 10 and 100 mM buffer solutions at the same pH. The decomposition of the assembly was rapid when the electrode potential was applied in pH 9 solutions, while the response was relatively slow in pH 10 and 11 solutions. All the results are rationalized on the basis of an electrochemically induced acidification of the local environment around the (avidin-ib-PEI)5 film on the Pt layer.     

H(2)-CO(2)-Dependent Anaerobic O-Demethylation Activity in Subsurface Sediments and by an Isolated Bacterium

The ability of microorganisms in sediments from the Atlantic Coastal Plain to biodegrade methoxylated aromatic compounds was examined. O-demethylation activity was detected in deep (121- and 406-m) sediments, as well as in the surface soil. A syringate-demethylating consortium, containing at least three types of bacteria, was enriched from a deep-sediment sample in a medium containing syringate as the sole organic carbon source and with a N(2)-CO(2) atmosphere. An isolate which demethylated syringate was obtained from the enrichment on an agar medium incubated under a H(2)-CO(2) but not a N(2)-CO(2) or N(2) atmosphere. O demethylation of syringate of this isolate was dependent on the presence of both H(2) and CO(2) in the gas phase. The metabolism of syringate occurred in a sequential manner: methylgallate accumulated transiently before it was converted to gallate. Mass balance analysis suggests that the stoichiometry of the reaction in this isolate proceeds in accordance with the following generalized equation: C(7)H(3)O(3)(OCH(3))(n) + nHCO(3) + nH(2) --> C(7)H(3)O(3)(OH)(n) + nCH(3)COO + nH(2)O.     

Effect of ventilation-perfusion inhomogeneity and N(2)O on oxygenation: physiological modeling of gas exchange.

Ventilation-perfusion (VA/Q) inhomogeneity was modeled to measure its effect on arterial oxygenation during maintenance-phase anesthesia involving an inspired mixture of 30% O(2) and either N(2)O or N(2). A multialveolar compartment computer model was constructed based on a log normal distribution of VA/Q inhomogeneity. Increasing the log SD of the distribution of blood flow from 0 to 1.75 produced a progressive fall in arterial PO(2) (Pa(O(2))). The fall was less steep in the presence of N(2)O than when N(2) was present instead. This was due mainly to the concentrating effect of N(2)O uptake on alveolar PO(2) in moderately low VA/Q compartments. The improvement in Pa(O(2)) when N(2)O was present instead of N(2) was greatest when the degree of VA/Q inhomogeneity was in the range typically seen in anesthetized patients. Models based on distributions of expired and inspired alveolar ventilation give quantitatively different results for Pa(O(2)). In the presence of VA/Q inhomogeneity, second-gas and concentrating effects may have clinically significant effects on arterial oxygenation even at "steady-state" levels of N(2)O uptake.     

T细胞亚群CD4测定方法的研究

为比较外周血T淋巴细胞亚群CD4不同测定方法的差别,以流式细胞术为定量手段,测定了猴外周血中三种不同方法处理后CD4的表达.结果表明：先标后溶法——先用异硫氰荧光素标记的单克隆抗体（FITC-CD4 McAb）标记后,再加入红细胞溶解液溶掉红细胞的处理方法,结果基本等同于传统的淋巴细胞分离法,但样本用量仅为传统方法的1/5,且操作简单.激光共焦显微术的形态学研究也证实：先标后溶法与淋巴细胞分离法相似,其细胞膜表面荧光标记清晰,优于先溶后标法.     

Effect of stage of the estrous cycle on interval to estrus after PGF(2alpha) in beef cattle

Effect of stage of the estrous cycle at the time of prostaglandin F(2alpha) (PGF(2alpha)) injection on subsequent reproductive events in beef females was studied in four trials involving 194 animals. Cycling animals were given two injections of 25 mg PGF(2alpha) 11 days apart or, in some cases, the interval was altered to allow the second injection to fall on a specific day of the cycle. Day of estrous cycle at time of the second injection was determined by estrous detection. Interval from the second PGF(2alpha) injection to the onset of estrus (interval to estrus) was shorter (P<.01) in heifers than in cows. Both cows and heifers injected on days 5 to 9 (early cycle) had a shorter (P<.01) interval to estrus (estrus = day 0) than did those injected on days 10 to 15 (late cycle). Conception rate was lower (P<.05) for early-cycle heifers than for late-cycle heifers inseminated by appointment at 80 hours. There was no significant difference in conception rate of early-or late-cycle heifers or cows inseminated according to estrous detection or early- or late-cycle cows inseminated at 80 hours. Progesterone concentrations in blood samples collected in heifers at 4-hour intervals after the second PGF(2alpha) injection on either day 7 or day 14 declined linearly (P<.05) through 36 hours. Day of the estrous cycle at PGF(2alpha) injection had no effect on rate of progesterone decline, even though heifers injected on day 7 had a shorter (P<.05) interval to estrus. All animals whose cycle length was not affected by the second PGF(2alpha) injection were treated on days 5 through 8 of the cycle, indicating that PGF(2alpha) was less effective in regressing the corpus luteum between days 4 and 9 of the cycle than later in the cycle.     

Immobilization of Burkholderia sp. lipase on a ferric silica nanocomposite for biodiesel production

In this work, lipase produced from an isolated strain Burkholderia sp. C20 was immobilized on magnetic nanoparticles to catalyze biodiesel synthesis. Core-shell nanoparticles were synthesized by coating Fe(3)O(4) core with silica shell. The nanoparticles treated with dimethyl octadecyl [3-(trimethoxysilyl) propyl] ammonium chloride were used as immobilization supporters. The Burkholderia lipase was then bound to the synthesized nanoparticles for immobilization. The protein binding efficiency on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 97%, while the efficiency was only 76% on non-modified Fe(3)O(4)-SiO(2). Maximum adsorption capacity of lipase on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 29.45 mg g(-1) based on Langmuir isotherm. The hydrolytic kinetics (using olive oil as substrate) of the lipase immobilized on alkyl-grafted Fe(3)O(4)-SiO(2) followed Michaelis-Menten model with a maximum reaction rate and a Michaelis constant of 6251 Ug(-1) and 3.65 mM, respectively. Physical and chemical properties of the nanoparticles and the immobilized lipase were characterized by Brunauer-Emmett-Teller (BET) analysis, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FT-IR). Moreover, the immobilized lipase was used to catalyze the transesterification of olive oil with methanol to produce fatty acid methyl esters (FAMEs), attaining a FAMEs conversion of over 90% within 30 h in batch operation when 11 wt% immobilized lipase was employed. The immobilized lipase could be used for ten cycles without significant loss in its transesterification activity.     

Mitochondrial beta-oxidation in Aspergillus nidulans

Beta-oxidation (beta-ox) occurs exclusively in the peroxisomes of Saccharomyces cerevisiae and other yeasts, leading to the supposition that fungi lack mitochondrial beta-ox. Here we present unequivocal evidence that the filamentous fungus Aspergillus nidulans houses both peroxisomal and mitochondrial beta-ox. While growth of a peroxisomal beta-ox disruption mutant (DeltafoxA) was eliminated on a very long-chain fatty acid (C(22:1)), growth was only partially impeded on a long-chain fatty acid (C(18:1)) and was not affected at all on short chain (C4-C6) fatty acids. In contrast, growth of a putative enoyl-CoA hydratase mutant (DeltaechA) was abolished on short-chain and severely restricted on long- and very long-chain fatty acids. Furthermore fatty acids inhibited growth of the DeltaechA mutant but not the DeltafoxA mutant in the presence of an alternate carbon source (lactose). Disruption of echA led to a 28-fold reduction in 2-butenoyl-CoA hydratase activity in a preparation of organelles. EchA was also required for growth on isoleucine and valine. The subcellular localization of the FoxA and EchA proteins was confirmed through the use of red and green fluorescent protein fusions.     

Formation of (R)-2,3-dihydrogeranylgeranoic acid from geranylgeraniol in rat thymocytes

In a metabolic study of [1-(14)C]geranylgeranial involving rat thymocytes, the radioactivity was mainly incorporated into two metabolites, Z1 and Z2, the latter moving slower than the former on a silica-gel thin-layer plate. The time course of Z1 and Z2 formation superficially suggested a precursor-product relationship between Z1 and Z2. The two metabolites were chemically converted to their methyl esters on treatment with trimethylsilyl diazomethane. Z1 was cochromatographed with E,E,E-geranylgeranoic acid (GGA). Z2 was prepared in a large quantity from geranylgeranial using thymocytes, and purified by TLC followed by ESI (negative ion mode) or EI mass-spectrometry. The observation of a negative ion at m/z 305 on ESI and a molecular ion at m/z 306 (C(20)H(34)O(2)) with fragments similar to GGA on EI implied that Z2 was dihydroGGA, which has been detected in the urine and serum of patients with Refsum disease. The EI mass spectrum of (R)-2,3-dihydroGGA was identical to that of Z2. The diastereomeric amide synthesized from metabolite Z2 with (R)-1-(1-naphtyl)ethylamine was cochromatographed with (R acid, R) amide, not with (S acid, R) amide, which were similarly synthesized from (R)- and (S)-2,3-dihydroGGAs, respectively. In another metabolic study on [1-(14)C]geranylgeraniol (GGOH), the radioactivity was similarly incorporated into a metabolite corresponding to (R)-2,3-dihydroGGA. (R)-2,3-DihydroGGA induced DNA ladder formation with a maximum at 15 mciroM in thymocytes. However, 2,3-dihydrofarnesoic acid did not induce it at all.     

Optimization of microalgal production in a shallow outdoor flume

The marine diatom Cyclotella cryptica was grown over a period of 13 months in a 48-m(2) shallow outdoor flume. The use of foil arrays at intervals of 1.2 m to effect systematic vertical mixing in the flume was found to significantly enhance microalgal production (p = 0.006). Average photosynthetic efficiencies (based on visible irradiance) with and without the foil arrays in place were 9.6 +/- 0.8 and 7.5 +/- 0.5% (+/-95% confidence intervals), respectively. A cost-benefit analysis indicated that the foil arrays were cost-effective if the value of the algae exceeded about $2.28 kg(1) of ash-free dry weight (AFDW). Parallel experiments performed in four 9.2-m(2) flumes showed that production was maximized when the cells were grown on a 2-day batch cycle between harvests rather than on a 1- or 3-day batch cycle. The optimum initial concentration (immediately after harvesting) of the algae was negatively correlated with the time interval between harvests and ranged from approximately 39 g AFDW/m(3) on a 3-day cycle to 213 g AFDW/m(3) on a 1-day cycle. The increase in production resulting from growth on a 2-day rather than a 1-day batch cycle was about 19% and was statistically significant at p = 0.0003. Growth of C. cryptica over a total period of 122 days during the 13-month study in the 48-m(2) flume under near-optimal conditions (2-day batch cycle, initial concentration 155 g AFDW/m(3)) resulted in an average production rate (+/-95% confidence interval) of 29.7 +/- 2.7 g AFDW/m(2) d.     

Eplerenone inhibits the intracrine and extracellular actions of angiotensin II on the inward calcium current in the failing heart. On the presence of an intracrine renin angiotensin aldosterone system

The influence of chronic administration of eplerenone on the intracrine as well as on the extracellular action of angiotensin II (Ang II) on L-type inward calcium current was investigated in the failing heart of cardiomyopathic hamsters (TO-2).For this, eplerenone (200 mg/kg/day) was administered orally to 2 month-old cardiomyopathic hamsters for a period of 3 months. Measurements of the peak inward calcium current (I(Ca)) was performed in single cells under voltage clamp using the whole cell configuration. The results indicated that eplerenone suppressed the intracrine action of Ang II (10(-)(8) M) on peak I(Ca) density. Moreover, the intracellular dialysis of the peptide did not change the time course of I(Ca) inactivation in animals treated chronically with eplerenone. The extracellular administration of Ang II (10(-)(8) M) incremented the peak I(Ca) density by only 20+/-8% (n=30) compared with 38+/-4% (n=35) (P<0.05) obtained in age-matched cardiomyopathic hamsters not exposed to eplerenone. Interestingly, the inhibitory of eplerenone (10(-7) M) on the intracrine action of Ang II was also found, in vitro, but required an incubation period of, at least, 24 h. The inhibitory action of eplerenone on the intracellular action of Ang II was partially reversed by exposing the eplerenone-treated cells to aldosterone (10 nM) for a period of 24 h what supports the view that: a) the mineralocorticoid receptor(MR) was involved in the modulation of the intracrine action of the peptide; b) the effect of eplerenone on the intracrine as well as on the extracellular action of Ang II was related ,in part, to a decreased expression of membrane-bound and intracellular AT1 receptors. In conclusion: a) eplerenone inhibits the intracrine action of Ang II on inward calcium current and reduces drastically the effect of extracellular Ang II on I(Ca); b) aldosterone is able to revert the effect of eplerenone; c) the mineralocorticoid receptor is an essential component of the intracrine renin angiotensin aldosterone system.     

Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep

The objective was to investigate the effects of dietary energy and urea supplementation on oocyte and embryo quality in sheep using in vivo and in vitro experimental models. Sixty-three ewes were fed grass meal at 0.5 or 2.0 times maintenance energy requirements (MER). The diet was supplemented with feed grade urea (U) for half of the ewes on each energy treatment. Ewes were stimulated with 1000 IU eCG and either slaughtered on the day of pessary withdrawal, for in vitro embryo production, or mated and slaughtered on Day 5 for embryo recovery. Urea decreased cleavage rate (48.3 vs 39.7%) and consequently blastocyst rate (41.6 vs 36.8%) but the differences were not significant. Oocytes from animals on 2.0 MER had a lower cleavage rate (54.9 vs 36.0%) and blastocyst yield (49.3 vs 31.4%) than those on 0.5 MER. However, there was an interaction between urea and energy for cleavage (P = 0.04) and blastocyst yield (P = 0.03) indicating a variable response to urea in the presence of high energy. This was manifested by a decrease in cleavage rate in the presence of urea and high energy (22%, 8 of 36), and a reduction in blastocyst development (19%, 7 of 36). When blastocyst development rate was expressed as a proportion of cleaved oocytes there was no difference between groups; in addition, there was no difference between groups in terms of blastocyst hatching rate (overall mean 66.1%) or blastocyst cell number on Day 8 (overall mean +/- SEM, 138.4 +/- 9.0, n=61). The effect of urea on cleavage rate in vivo was more severe. Urea supplementation reduced (P<0.001) the cleavage rate (93 vs 62%). Despite this, the yield of blastocysts was unaffected. Oocytes from ewes on 0.5 MER exhibited a lower (P<0.05) cleavage rate than those on 2.0 MER (66 vs 87%). This effect was also apparent at the blastocyst stage (40.0 vs 50.9%), although the difference was no longer significant. There were no differences in hatching rate (overall mean 70.7%) or blastocyst cell numbers (overall mean +/- SEM, 166.3 +/- 15.6, n=40). Collectively, these results suggest that both high dietary energy and urea content influence subsequent embryo development in vitro, and the deleterious effects of urea are likely influenced by concomitant energy intake.     

Mobility of the conceptus and uterine contractions in the mare

Location of the embryonic vesicle within the uterus of mares was recorded every. five minutes for two consecutive hours (25 location determinations per trial) in three experiments. In Experiment 1 (n=7), the number of location changes among nine uterine segments (three body segments and three segments for each horn) was greater (P<0.05) on Day 13 than on Day 10. The vesicle was located in the body more frequently (P<0.05) and tended (P<0.1) to move to a more caudal position more frequently on Day 10 than on Day 13. Fixation occurred on Day 15 in four of seven mares and on Day 16 in the remaining three mares. The number of location changes was not significantly different between two days prior to fixation and one day prior to fixation. In Experiment 2, the effect of clenbuterol, a B₂ sympathomimetic blocker of uterine contractions, was studied on Days 12 or 13 of pregnancy. Location changes occurred less frequently (P<0.05) in treated mares (n=9) than in controls (n=10), indicating involvement of uterine contractions in the mobility of the embryonic vesicle. In Experiment 3, when the initial direction of location changes was caudal within a horn and cranial within the uterine body, the vesicle was more likely (P<0.05) to continue moving in the same direction than in the opposite direction. However, when the direction within a horn was cranial, the next location change was as likely to be in the opposite direction as in the same direction (not significantly different from equality). When the direction within the uterine body was caudal, the next location change was more likely (P<0.05) to be in the opposite direction.     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

The embryo influences adenylate cyclase activity and hormonal response in rabbit myometrium during early pregnancy

The effect of pseudopregnancy (PSPG; days: 0 (estrus), 1, 6.5, 9 and 15) and pregnancy (PG; days: 6.5, 9 and 15) on adenylate cyclase (AC) activity was verified in rabbit myometrium. During PSPG, there was a time related decline in basal activity from 71 +/- 16.2 (D 0) to 13.1 +/- 1.6 (PSPG-D9) pmoles cAMP formed/mg prot-min. Stimulation of the enzyme by GTP, Isoproterenol (ISO), Prostaglandin E2 (PGE2) or Sodium Fluoride (NaF) followed a similar pattern. AC activity was compared in myometrial tissues of pregnant animals (PG) separated into embryonic (ES) and interembryonic (IES) sites. On days 6.5 and 9, AC activity measured in tissues from PG rabbits (ES and IES) was always higher than that found in tissues from PSPG animals on corresponding days. On day 6.5, AC activity was slightly higher (p less than 0.01) in ES than in IES. This was confirmed on day 9 where basal as well as GTP, ISO and PGE2 stimulated activities were higher in ES than in IES (p less than 0.001). Dose response to ISO, expressed as % of GTP, were similar on D0, 1, 6.5 and 15 of PSPG. However, on day 9, there was a striking diminution in response reflected by a lower stimulation at suboptimal dose (0.1 microM; p less than 0.05) from 115 +/- 2 on day 0 to 104 +/- 4 on day 9. These results suggest that protein content which is increased during pseudopregnancy could be responsible for the decline of AC activity. However, results obtained on day 9 and 15 suggest that other factors are involved. Dose responses to ISO during PG showed an alteration in response on days 6.5 and 9 at ES. It was reflected by a higher stimulation at suboptimal (0.1 microM) and optimal doses (100 microM). These results suggest that myometrial AC activity could be regulated by the presence of the embryo.     

Effect of dioxane on the structure and hydration-dehydration of alpha-chymotrypsin as measured by FTIR spectroscopy

A new experimental approach based on FTIR spectroscopic measurements was proposed to study simultaneously the adsorption/desorption of water and organic solvent on solid enzyme and corresponding changes in the enzyme secondary structure in the water activity range from 0 to 1.0 at 25 degrees C. The effect of dioxane on the hydration/dehydration and structure of bovine pancreatic alpha-chymotrypsin (CT) was characterized by means of this approach. Dioxane sorption exhibits pronounced hysteresis. No sorbed dioxane was observed at low water activities (a(w)<0.5) during hydration. At a(w) about 0.5, a sharp increase in the amount of sorbed dioxane was observed. Dioxane sorption isotherm obtained during dehydration resembles a smooth curve. In this case, CT binds about 150 mol dioxane/mol enzyme at the lowest water activities. Three different effects of dioxane on the water binding by the initially dried CT were observed. At a(w)<0.5, water adsorption is similar in the presence and absence of dioxane. It was concluded that the presence of dioxane has little effect on the interaction between enzyme and tightly bound water at low a(w). At a(w)>0.5, dioxane increases the amount of water bound by CT during hydration. This behavior was interpreted as a dioxane-assisted effect on water binding. Upon dehydration at low water activities, dioxane decreases the water content at a given a(w). This behavior suggests that the suppression in the uptake of water during dehydration may be due to a competition for water-binding sites on chymotrypsin by dioxane. Changes in the secondary structure of CT were determined from infrared spectra by analyzing the structure of amide I band. Dioxane induced a strong band at 1628 cm(-1) that was assigned to the intermolecular beta-sheet aggregation. Changes in the intensity of the 1628 cm(-1) band agree well with changes in the dioxane sorption by CT. An explanation of the dioxane effect on the CT hydration and structure was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the solid enzyme. The reported results demonstrate that the hydration and structure of alpha-chymotrypsin depend markedly on how enzyme has been hydrated - whether in the presence or in the absence of organic solvent. A qualitative model was proposed to classify the effect of hydration history on the enzyme activity-a(w) profiles.     

Bioassay-guided isolation of an alkaloid with antiangiogenic and antitumor activities from the extract of Fissistigma cavaleriei root

Fissistigma cavaleriei (Levl) Rehd (Annonaceae) is used as a folklore medicine for treatment of inflammation, arthritis, and tuberculosis by Miao people in China. In the present study, the antiangiogenic activity of F. cavaleriei was investigated. The chorioallantoic membrane of the fertilized hen's egg (CAM assay) was used to determine antiangiogenic activity of the plant extract. Compound (1), a compound with antiangiogenic activity, was isolated by bioassay-guided fractionation from F. cavaleriei for the first time. The structure of compound (1) was elucidated on the basis of spectroscopic methods. Colorimetric COX (ovine) inhibitor screening assay was used to determine its inhibitory effect on COX-1 and COX-2. MTT and Sulforhodamine B assays were used to investigate its cytotoxic effects on tumor cell lines. As a result, compound (1) showed a selectively inhibiting effect on COX-2 and could inhibit the growth of tumor cells in vitro. The antitumor activity of compound (1) was further confirmed by the observation that compound (1) administration significantly inhibited the growth of S-180 cells in mice. Moreover, compound (1) was able to enhance the antitumor activity of doxorubicin in the mice bearing with S-180 cells while combined with doxorubicin. In conclusion, compound (1) is a multi-target molecule and further experimental investigations are needed to determine whether it can be used as a lead molecule for tumor treatment.     

Investigation of dual-layer membrane cloaking method by surface plasmon resonance for direct chronoamperometric immunoassay of serum sample

A "dual-layer membrane cloaking" (DLMC) method was developed to construct disposable electrochemical immunosensor for direct determination of serum sample. Mouse IgG (MIgG) molecules were firstly immobilized on a substrate. After the formation of a didodecyldimethylammonium bromide (DDAB) membrane on the MIgG modified substrate, an additional bovine serum albumin (BSA) thin layer was formed to build a BSA/DDAB dual-layer membrane (DLM). When alkaline phosphatase conjugated anti-mouse IgG antibodies (anti-MIgG-ALP) in human serum were incubated on the substrate, anti-MIgG-ALP was recognized specifically by the immobilized MIgG while all nonspecifically adsorbed proteins were selectively removed together with BSA/DDAB DLM by 5% Triton X-100 (v/v) before final measurements. The BSA/DDAB DLM was characterized and optimized by surface plasmon resonance (SPR) technique, and further employed in a disposable immunoassay based on an ITO chip. Under optimal conditions, MIgG in human serum was directly detected in the range of 2.0-18.0 ng mL(-1) without dilution or separation. A limit of detection as low as 0.922 ng mL(-1) (6.15 pM) was obtained. The proposed DLMC method can efficiently prevent the penetration of matrix proteins through single cloaking membrane and completely eliminate nonspecific adsorption. It has great potential in providing a versatile way for direct determination of serum sample with ultra-sensitivity.     

Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.     

Selective biorecognition and preservation of cell function on carbohydrate-immobilized phosphorylcholine polymers

To obtain synthetic materials capable of selectively recognizing proteins and cells, and preserving their functions, biomembrane mimetic polymers having a phospholipid polar group and carbohydrate side chains were designed. Poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-2-lactobionamidoethyl methacrylate (LAMA)] (PMBL) was synthesized and coated on substrates by solvent evaporation. Selective binding of galactose-recognized lectin, RCA120, to a PMBL surface was investigated by measurement of surface plasmon resonance. The binding of RCA120 to the PMBL surface was confirmed by a remarkable change in resonance angle. The apparent affinity constant of RCA120 to PMBL3.0 (3.0 mol % LAMA unit in the feed) per LAMA unit was 2.77 x 10(5) M(-1). When a glucose-recognized lectin, concanavalin A, was in contact with PMBL, no change in the resonance angle was observed, and any nonspecific fouling of protein on PMBL was effectively reduced. Cells of the human hepatocellular liver carcinoma cell line (HepG2) having asialoglycoprotein receptors (ASGPRs) were seeded on polymer surfaces. On poly(BMA) (PBMA), many adherent cells were observed and were well-spread with monolayer adhesion, but cell adhesion was reduced on poly(MPC-co-BMA) (PMB). HepG2 adhesion was observed on PMBL because the cell has ASGPRs; the number of cells adhering to the PMBL polymer surfaces increased with an increase in the density of galactose residues on the surface. In contrast, adhesion of NIH-3T3 cells to PMBL was reduced in a manner similar to that on PMB because the NIH-3T3 cells did not have ASGPRs. Cell adhesion to the PMBL surface was well-regulated by ligand-receptor interactions. Furthermore, some of the cells adhering to the PMBL surface had a spheroid form, and similarly shaped spheroids were scattered on the surface. Although poly(BMA-co-LAMA) (PBL) has galactose residues, the adherent cells were spread in a manner similar to those on PBMA. The MPC units in PMBL contribute to make a spheroid formation of HepG2 cells. The amount of albumin secreted from a cell was compared with the chemical structure of the substrate. The spheroid shaped cells cultured on the PMBL surface secreted much more albumin than did the spreading cells that adhered to the PBMA. In conclusion, the biomembrane mimetic carbohydrate-immobilized phosphorylcholine polymers produced a suitable surface for biorecognition and preservation of cell function.