The lipophilic fluorescent probe 4-dimethylaminochalcone: factors responsible for the fluorescence yield

Factors responsible for fluorescence quenching of the lipophylic fluorescent probe 4-dimethylaminochalcone in nonpolar and polar media were studied. The femtosecond dynamics of 4-dimethylaminochalcone excited state was measured using the absorption method of "excitation probing". In nonpolar hexane where the fluorescence quantum yield is very low (0.001), all excited 4-dimethylaminochalcone molecules go to the triplet state with a rate constant of 2.10(11) s(-1). At the same time, the radiation rate constant is 1000 times lower; therefore, such a fast transition to triplet is the major cause of the very small fluorescence yield. In polar acetone, the fluorescence yield is 220 times higher than in hexane. In acetone, no transitions to triplet state were detected. At the same time, a radiationless conversion to the ground state took place with a rate constant of 10(9) s(-1), which decreased the fluorescence yield to 0.22. The activation energy of the quenching processes is polarity dependent and decreases from 6 in nonpolar to 3 kcal/mol in polar media. The yield of 4-dimethylaminochalcone fluorescence varies hundreds times in media of different polarity but is a linear function of the Lippert's polarity parameter f(epsilon,n) where epsilon is the dielectric constant at low frequencies. It is supposed that polar media stabilize the "flat" conformation of the 4-dimethylaminochalcone molecule prior to its excitation, and this conformation is optimal for fluorescence. In this case, stabilization is determined only by medium polarity.     

Induction of cytoplasmic polarity in heterokaryons of mouse 4-cell-stage blastomeres fused with 8-cell- and 16-cell-stage blastomeres

Cell surface and cytoplasmic polarity is exhibited by the blastomeres of mouse preimplantation embryos following compaction at the 8-cell stage of cleavage. It has been hypothesized that cytoplasmic polarity is initiated by plasma membrane functions of polar blastomeres that are absent from apolar blastomeres. To test this hypothesis the plasma membranes of "test" polar and apolar 8-cell- and 16-cell-stage blastomeres were inserted into the plasma membrane of "carrier" 4-cell-stage blastomeres by polyethylene glycol-mediated fusion of carrier-test blastomere pairs. After a 4-hr culture period each heterokaryon was scored for the distribution of two marker organelles--lipid droplets and nuclei--with respect to their proximity to the plasma membrane insert from the test blastomere. Plasma membrane inserts from polar test blastomeres were identified by labeling their apical domains with fluorescently tagged (succinylated) concanavalin A. The incidence of polar heterokaryons (those exhibiting a discrete fluorescently labeled area of plasma membrane corresponding to the apical domain inherited from the test blastomere) was 55/85 (69%) and 48/79 (61%) for 8-cell-stage and 16-cell-stage test blastomeres, respectively. In all polar heterokaryons, both nuclei were subjacent to the fluorescent label (apical domain of a polar plasma membrane insert), while the majority of lipid droplets resided in the hemisphere opposite the fluorescent label. In all 61 apolar heterokaryons examined (those lacking a discrete fluorescently labeled plasma membrane area) both nuclei were centrally located and lipid droplets were randomly distributed. These observations are consistent with the hypothesis that cytoplasmic polarity can be initiated by properties that distinguish the plasma membranes of polar blastomeres from those of apolar blastomeres.     

Effect of non aqueous solvent on structural stability of α-amylase: A cost-effective prospective for protein stabilization

The aim of the present study is to assess the effect of non-aqueous organic solvent on structural stability, molecular integrity and structure of α-amylase. The activity and thermal stability of the enzyme was measured before and after treatment with non polar solvent (i.e. hexane). The activity was found to be marginally affected and thermal stability was found to be significantly increased after treatment with hexane. The enzyme was found to be more resistant to thermal inactivation in hexane compared to in an aqueous buffer. The fluorescence measurement indicated a blue shift of 3 nm in the emission maximum (λ_max) probably due to a minor change in the polarity of aromatic amino acid residues after treatment with a non-aqueous solvent. Assessment of thermal denaturation profile, 1-anilino-8-naphthalene-sulfonate (ANS) binding and acrylamide quenching of the enzyme suggested an increase in the molecular integrity and overall stability of the enzyme after treatment with hexane. However, these entire molecular events were not accompanied by any major change in the secondary structure. Our findings suggest that treatment of proteins or enzymes in non-aqueous solvents could be an attractive and cost-effective strategy to improve their structural stability without compromising their biological functions.     

Polarity of the mouse embryo is anticipated before implantation

In most species, the polarity of an embryo underlies the future body plan and is determined from that of the zygote. However, mammals are thought to be an exception to this; in the mouse, polarity is generally thought to develop significantly later, only after implantation. It has not been possible, however, to relate the polarity of the preimplantation mouse embryo to that of the later conceptus due to the lack of markers that endure long enough to follow lineages through implantation. To test whether early developmental events could provide cues that predict the axes of the postimplantation embryo, we have used the strategy of injecting mRNA encoding an enduring marker to trace the progeny of inner cell mass cells into the postimplantation visceral endoderm. This tissue, although it has an extraembryonic fate, plays a role in axis determination in adjacent embryonic tissue. We found that visceral endoderm cells that originated near the polar body (a marker of the blastocyst axis of symmetry) generally became distal as the egg cylinder formed, while those that originated opposite the polar body tended to become proximal. It follows that, in normal development, bilateral symmetry of the mouse blastocyst anticipates the polarity of the later conceptus. Moreover, our results show that transformation of the blastocyst axis of symmetry into the axes of the postimplantation conceptus involves asymmetric visceral endoderm cell movement. Therefore, even if the definitive axes of the mouse embryo become irreversibly established only after implantation, this polarity can be traced back to events before implantation.     

Cell polarity signaling in Arabidopsis involves a BFA-sensitive auxin influx pathway

Coordination of cell and tissue polarity commonly involves directional signaling. In the Arabidopsis root epidermis, cell polarity is revealed by basal, root tip-oriented, hair outgrowth from hair-forming cells (trichoblasts). The plant hormone auxin displays polar movements and accumulates at maximum concentration in the root tip. The application of polar auxin transport inhibitors evokes changes in trichoblast polarity only at high concentrations and after long-term application. Thus, it remains open whether components of the auxin transport machinery mediate establishment of trichoblast polarity. Here we report that the presumptive auxin influx carrier AUX1 contributes to apical-basal hair cell polarity. AUX1 function is required for polarity changes induced by exogenous application of the auxin 2,4-D, a preferential influx carrier substrate. Similar to aux1 mutants, the vesicle trafficking inhibitor brefeldin A (BFA) interferes with polar hair initiation, and AUX1 function is required for BFA-mediated polarity changes. Consistently, BFA inhibits membrane trafficking of AUX1, trichoblast hyperpolarization induced by 2,4-D, and alters the distal auxin maximum. Our results identify AUX1 as one component of a novel BFA-sensitive auxin transport pathway polarizing cells toward a hormone maximum.     

Optimizing lipase activity, enantioselectivity, and stability with medium engineering and immobilization for beta-blocker synthesis.

Lipase from Pseudomonas cepacia showed poor activity and moderate enantioselectivity (E) in pure aqueous systems for hydrolysis of a racemic mixture (+/-)-1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane, which is a potential intermediate for beta-blocker synthesis. However, addition of polar organic solvents to the reaction can change both the activity and the enantioselectivity for this chiral reaction significantly. It was observed, in general, that the activity increases and the enantioselectivity decreases with the increase in the polarity of the organic solvent added to the medium. Among the six solvents chosen (i.e., dimethylsulfoxide [DMSO], 1, 4-dioxane, dimethylformamide [DMF], acetone, 1-propanol, and tetrahydrofuran [THF]), maximum activity and minimum enantioselectivity was obtained with DMSO, whereas minimum activity and maximum enantioselectivity was obtained with THF as the cosolvents. In the subsequent studies, native or polyethylene glycol (PEG)-modified lipase was immobilized by entrapping in Caalginate gel beads. In a fixed-bed continuous reactor containing these catalyst beads, the enzyme was found to be at least three times more enantioselective than the native form in a batch reactor. This fixed-bed reactor with the beads could be operated with high concentration of acetone (33% v/v) for about 1 month without a significant loss of enzyme activity and enantioselectivity.     

Risk predictive model for damage caused by the spittlebug Aeneolamiapostica (Walker) Fennah (Hemiptera: Cercopidae)

This paper evaluated the risk that Aenolamia postica (Walker) Fennah populations reach the economic threshold in sugar cane fields in Veracruz, México. A risk deductive model was constructed to include the sequence of events leading to damaging populations, considered the top event or critical failure in the crop. Model events were identified and quantified, and model was validated on field conditions. The model components and their state values were identified as: temperature e" 28 degrees C, precipitation e" 45% during June and July, soil clay content e" 40%, infested adjoining fields, deficient weed control, wind dominance, crop phenology and variety, deficient chemical and biological control, and irrigation. Sensitivity analysis showed that the most important events triggering high densities of A. postica were high temperatures and precipitation, previous field infestation, nymph and weed presence. Event probability estimates were combined using Boolean algebra to compute the minimum, mean and maximum probabilities for the top event, yielding values of 0.417, 0.563, y 0.734 respectively. Model was tested in field, by selecting sugar cane fields having the model properties and compared to fields without these features. Fields were sampled in both conditions during 2004 year and high-risk fields had significantly (F = 13, 4, gl = 1, 18, P = 0,0018) higher densities (2.4 adults m(-1)) than low-risk plots (0.4 adults m(-1)) thus agreeing with the model forecast.     

Influence of 2,3,5-Triiodobenzoic Acid and 1-N-Naphthylphthalamic Acid on Indoleacetic Acid Transport in Carnation Cuttings: Relationship with Rooting

³H-IAA transport in excised sections of carnation cuttings was studied by using two receiver systems for recovery of transported radioactivity: agar blocks (A) and wells containing a buffer solution (B). When receivers were periodically renewed, transport continued for up to 8 h and ceased before 24 h. If receivers were not renewed, IAA transport decreased drastically due to immobilization in the base of the sections. TIBA was as effective as NPA in inhibiting the basipetal transport irrespective of the application site (the basal or the apical side of sections). The polarity of IAA transport was determined by measuring the polar ratio (basipetal/acropetal) and the inhibition caused by TIBA or NPA. The polar ratio varied with receiver, whereas the inhibition by TIBA or NPA was similar. Distribution of immobilized radioactivity along the sections after a transport period of 24 h showed that the application of TIBA to the apical side or NPA to the basal side of sections, increased the radioactivity in zones further from the application site, which agrees with a basipetal and acropetal movement of TIBA and NPA, respectively. The existence of a slow acropetal movement of the inhibitor was confirmed by using ³H-NPA. From the results obtained, a methodological approach is proposed to measure the variations in polar auxin transport. This method was used to investigate whether the variations in rooting observed during the cold storage of cuttings might be related to changes in polar auxin transport. As the storage period increased, a decrease in intensity and polarity of auxin transport occurred, which was accompanied by a delay in the formation and growth of adventitious roots, confirming the involvement of polar auxin transport in supplying the auxin for rooting. Received April 19, 1999; accepted December 2, 1999     

The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA

Guanine auxotrophs of Escherichia coli K-12 were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or the acridine mustard ICR 372. guaA (xanthosine 5'-monophosphate [XMP] aminase-less) mutants were distinguished from guaB (inosine 5'-monophosphate [IMP] dehydrogenase-less) mutants by their growth response to xanthine and by enzyme assay. Mutations were classified as base substitutions or frameshift on the basis of mutagen-induced reversion patterns. All guaA strains, including three frameshift mutants, produced derepressed levels of IMP dehydrogenase when cultured with a growth-limiting concentration of guanine. The guaB strains were of two types: (i) those producing derepressed levels of XMP aminase, and (ii) those producing basal levels of XMP aminase when grown under conditions of guanine starvation. In the guaB strains of the second type, the expression of the adjacent guaA gene is reduced. It is proposed that this pleiotropic effect of some guaB mutations is a result of polarity. The orientation of polarity suggests the gene order "operator"-guaB-guaA. Gel diffusion studies with IMP dehydrogenase antiserum showed that strains carrying polar guaB mutations do not produce cross-reacting material (CRM). The remaining guaB mutants were either CRM(+) or CRM(-). Mapping the mutations by three-factor crosses showed that polar and nonpolar guaB sites are clustered in a small genetic region cotransducible with guaA. The relative positions of the guaB mutational sites established that the polar mutations lie within the structural gene for IMP dehydrogenase.     

Mangrove resilience to climate extreme events in a Colombian Caribbean Island

Extreme events affect coastal vegetation in several ways. They cause massive tree defoliations and mortality that carry changes in vegetation structure and floristic composition. In order to assess these changes in dry tropical forests, coastal vegetation and mangroves in El Rosario archipelago, and their relationship with extreme events between 2002 and 2014, Quickbird and Worldview Satellite images with uneven periodicity were analyzed and the Normalized Difference Vegetation Index (NDVI) was calculated for these vegetation covers. Time series analysis was performed to oceanographic and climate variables such as maximum wind velocity, daily precipitation, significant wave height, peak wave period and maximum and minimum air temperatures. The first two axes of a redundancy analysis explained 65% of data variance (p value ≤ 0.05) and showed that the decrease of the NDVI and extension of beach vegetation were related to increases in wind frequency and intensity. However, mangrove vegetation was benefited by the increase in the frequency of short drought events, although their NDVI decreased when these drought events became longer. Drought events were related to El Niño Southern Oscillation (ENSO) and Pacific Decadal Oscillation (PDO) anomalies that had the strongest effects on coastal vegetation and dry forest, as shown by the minimum values of NDVI recorded in 2010. Mangrove vegetation was more resilient to such droughts and strong winds than coastal and dry forest vegetation due to their location along bays or surrounding internal lagoons.     

Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup

As part of the Fourth International Workshop on Genotoxicity Testing (IWGT), held 9-10 September 2005 in San Francisco, California, an expert working group on the Comet assay was convened to review and discuss some of the procedures and methods recommended in previous documents. Particular attention was directed at the in vivo rodent, alkaline (pH >13) version of the assay. The aim was to review those protocol areas which were unclear or which required more detail in order to produce a standardized protocol with maximum acceptability by international regulatory agencies. The areas covered were: number of dose levels required, cell isolation techniques, measures of cytotoxicity, scoring of comets (i.e., manually or by image analysis), and the need for historical negative/positive control data. It was decided that a single limit dose was not sufficient although the required number of dose levels was not stipulated. The method of isolating cells was thought not to have a qualitative effect on the assay but more data were needed before a conclusion could be drawn. Concurrent measures of cytotoxicity were required with histopathological examination of tissues for necrosis or apoptosis as the "Gold Standard". As for analysing the comets, the consensus was that image analysis was preferred but not required. Finally, the minimal number of studies required to generate a historical positive or negative control database was not defined; rather the emphasis was placed on demonstrating the stability of the negative/positive control data. It was also agreed that a minimum reporting standard would be developed which would be consistent with OECD in vivo genotoxicity test method guidelines.     

灵芝子实体中不同极性的三萜体外抗肿瘤及抗炎活性比较

为了解灵芝中不同极性三萜活性的差异,运用大孔树脂将灵芝总三萜根据极性大小进行分段,采用高效液相法分析各极性部分的HPLC指纹图谱和三萜含量,并比较其对不同肿瘤细胞增殖的抑制作用和抗炎活性。结果显示D-101大孔树脂可以有效地将灵芝总三萜分为中等极性和低极性两部分,两部分的三萜含量分别为(279.00±2.90)mg/g、(94.52±2.03)mg/g。其中,低极性三萜的体外抗肿瘤活性明显强于中等极性三萜,其对K562、SW620、L1210细胞增殖抑制的IC₅₀值分别为(74.12±1.94)μg/mL、(121.45±2.13)μg/mL、(13.52±1.13)μg/mL。另外,低极性三萜对RAW264.7细胞呼吸爆发的抑制作用也明显强于中等极性三萜。进一步对低极性三萜和中等极性三萜单体的抗炎活性进行比较,发现低极性三萜灵芝烯酸F、灵芝酸DM、灵芝醇B对RAW264.7细胞呼吸爆发的抑制作用明显强于极性较高的灵芝酸C₂、灵芝酸A。该研究阐明了灵芝子实体中不同极性三萜部位的抗肿瘤及抗炎活性并不相同,为将来新药开发和灵芝质量标准的改进建立了基础。     

The kinetics of cytological events during double fertilization in Zea mays L.

By using a clearing method, the process of double fertilization in Zea mays L. (line A 188) was analysed and the precise sequence of events was determined. The period from pollen tube arrival to gamete fusion was relatively short, possibly less than 1 h. The karyogamy was of premitotic type, and the time from the contact of male and female nuclei to the fusion of male and female nucleoli was about 5 h in the egg cell and 3 h in the central cell. In the central cell, the sperm nucleus fused with either one of the polar nuclei or the secondary nucleus, the latter being observed for the first time in maize. The zygote was in the resting period for 13–16 h before division commenced, changing the cell polarity during karyogamy and the resting period. The primary endosperm nucleus divided immediately after karyogamy was completed in the central cell. The embryo sacs with two-celled proembryos contained four to eight endosperm nuclei. The timetable of fertilization events could be a standard for further studies on in vitro fertilization at the cytological and molecular levels.     

Intramembrane events during the nerve impulse

In current literature the cell adhesion to solid surfaces has been treated in the context of basic physicochemical forces. However, in all these reports the concept of solid surface force has not been properly analyzed. The surface forces acting across an interface formed when two phases meet has been shown to consist of dispersion (attraction) forces and polar forces (arising from different interactions). Current theories have repeatedly neglected the role of polar forces in the cell adhesion. In order to clarify this concept, the particular case, i.e. adhesion of cells on polystyrene surfaces with varying degree of polar groups is described. In this case, the adhesion of cells was reported to increase with polarity of polystyrene, and this agrees with the present study that the solid polar force component increased in the same manner.     

Using SCC8, SCF27 and VMC7f2 markers in grapevine breeding for seedlessness via marker assisted selection

We used molecular markers associated with seedlessness in grapes, namely SCC8, SCF27 and VMC7f2, to improve the efficiency of seedless grapevine breeding via marker assisted selection (MAS). DNA from 372 F(1) hybrid progeny from the cross between seeded "Alphonse Lavallée" and seedless "Sultani" was amplified by PCR using three markers. After digestion of SCC8 marker amplification products by restriction enzyme BgIII, 40 individuals showed homozygous SCC8+/SCC8+ alleles at the seed development inhibitor (SdI) locus. DNA from 80 of the progeny amplified with the SCF27 marker produced bands; 174 individuals had 198-bp alleles of the VMC7f2 marker associated with seedlessness. In the second year, based on MAS, 183 F(1) hybrids were designated as seedless grapevine candidates because they were positive for a minimum of one marker. Twenty individuals were selected as genetic resources for future studies on seedless grapevine breeding because they carried alleles for the three markers associated with seedlessness. The VMC7f2 SSR marker was identified as the marker most associated with seedlessness.     

Cytodifferentiation of polar plant cells Use of anti-microtubular agents during the differentiation of statocytes from cress roots (Lepidium sativum L.)

The development of the structural polarity of statocytes from cress roots (Lepidium sativum L.) was studied in a time- and stage-dependent manner. Outgrowing radicles had statocytes with abundant lipid droplets, sparsely developed endoplasmic reticulum (ER) and nuclei located at the proximal cell poles. During differentiation, coincidentally the lipid droplets disappeared, while rough ER increased in length. The ER was translocated into the distal cell pole to establish a complex of stacked ER. Microtubules occurred first at the distal cell edges. As a second step, ER was produced in the vicinity of the nucleus and was also translocated distally. By application of the antimicrotubular agents heavy water (90%), colchicine (10^-4 mol·l^-1) and triethyl lead chloride (20 mol·l^-1), the involvement of microtubules in these events was studied. Triethyl lead chloride led to a complete cessation of differentiation; root-cap cells remained at a stage without polar arrangement of the ER. Colchicine affected the development of structural polarity slightly, as shown by a higher density of cortical ER cisternae. Heavy water inhibited the translocation of ER almost completely and yielded ER located also in the cell center. All anti-microtubular agents inhibited cell division and the differentiation of the distal cell layer of the dermatocalyptrogen into statocytes. It is hypothesized that microtubules serve as anchoring sites for microfilaments, which actually mediate the translocation of the ER. Hence, an intact system of microtubules and microfilaments is necessary for the expression of structural polarity.Abbreviations DC dermatocalyptrogen - ER endoplasmic reticulum - M meristem cell layer - MT microtubule - pI prospective story I - TrEl triethyl lead chloride     

4‐alkoxyethoxy‐N‐octadecyl‐1,8‐naphthalimide fluorescent sensor for human serum albumin and other major blood proteins: design,synthesis and solvent effect

A series of 4‐alkoxyethoxy‐N‐octadecyl‐1,8‐naphthalimides with intense blue fluorescence were designed and synthesized as polarity and spectrofluorimetric probes for the determination of proteins. In solvents of different polarities, the Stokes shifts of two dyes increased with increasing solvent polarity and fluorescence quantum yields decreased significantly, suggesting that electronic transiting from ground to excited states was π–π^* in character. Dipole moment changes were estimated from solvent‐dependent Stokes shift data using a solvatochromic method based on bulk solvent polarity functions and the microscopic solvent polarity parameter (). These results were generally consistent with semi‐empirical molecular orbital calculations and were found to be quite reliable based on the fact that the correlation of the solvatochromic Stokes shifts with was superior to that obtained using bulk solvent polarity functions. Fluorescence data revealed that the fluorescence quenching of human serum albumin (HSA) by dyes was the result of the formation of a Dye–HSA complex. The method was applied to the determination of total proteins (HSA + immunoglobulins) in human serum samples and results were in good agreement with those reported by the research institute. Copyright © 2012 John Wiley & Sons, Ltd.     

Thermal biology and immersion tolerance of the Beringian pseudoscorpion <Emphasis Type="Italic">Wyochernes asiaticus</Emphasis>

Wyochernes asiaticus (Arachnida: Pseudoscorpiones: Chernetidae) is a pseudoscorpion distributed across Beringia, the areas of Yukon, Alaska and Siberia that remained unglaciated at the last glacial maximum. Along with low temperatures, its streamside habitat suggests that submergence during flood events is an important physiological challenge for this species. We collected W. asiaticus in midsummer from 66.8°N Yukon Territory, Canada, and measured thermal and immersion tolerance. Wyochernes asiaticus is freeze-avoidant, with a mean supercooling point of ?6.9 °C. It remains active at low temperatures (mean critical thermal minimum, CT_min, is ?3.6 °C) and has a critical thermal maximum (CT_max) of 37.8 °C, which is lower than other arachnids and consistent with its restriction to high latitudes. Fifty per cent of W. asiaticus individuals survived immersion in oxygen-depleted water for 17 days, suggesting that this species has high tolerance to immersion during flooding events. To our knowledge, these are the first data on the environmental physiology of any pseudoscorpion and a new addition to our understanding of the biology of polar microarthropods.     

maelstrom is required to position the MTOC in stage 2–6 Drosophila oocytes

The factors that determine intracellular polarity are largely unknown. In Drosophila oocytes one of the earliest polar events is the positioning of the microtubule-organizing center (MTOC). Here we present data that are consistent with the hypothesis that maelstrom is required for posterior positioning of the MTOC.     

Mechanical properties of the surface of the sea urchin egg at fertilization and during cleavage

1. 1. Changes in stiffness of the cell surface at fertilization and during cleavage in sea urchin eggs were determined by the magnetic particle method.

2. 2. The stiffness of the cell surface increased at fertilization, reached a maximum after about 1.5 min, then decreased and reached a minimum about 4 min after insemination, followed by a gradual increase, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5 °C.

3. 3. The stiffness of the cell surface increased during the diaster stage, reached a maximum 2 to 3 min before the onset of cleavage, then decreased to a minimum about 1 min before the onset of cleavage, increased again, reached a maximum during cleavage and then diminished, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5 °C. A similar stiffness change was observed in the eggs of Hemicentrotus pulcherrimus at 17 to 19 °C, occurring almost in parallel in both the equatorial and polar surfaces.