EIN3-like gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Grande naine)

Ethylene signal transduction initiates with ethylene binding at receptor proteins and terminates in a transcription cascade involving the EIN3/EIL transcription factors. Here, we have isolated four cDNAs homologs of the Arabidopsis EIN3/EIN3-like gene, MA-EILs (Musa acuminata ethylene insensitive 3-like) from banana fruit. Sequence comparison with other banana EIL gene already registered in the database led us to conclude that, at this day, at least five different genes namely MA-EIL1, MA-EIL2/AB266318, MA-EIL3/AB266319, MA-EIL4/AB266320 and AB266321 exist in banana. Phylogenetic analyses included all banana EIL genes within a same cluster consisting of rice OsEILs, a monocotyledonous plant as banana. However, MA-EIL1, MA-EIL2/AB266318, MA-EIL4/AB266320 and AB266321 on one side, and MA-EIL3/AB266319 on the other side, belong to two distant subclusters. MA-EIL mRNAs were detected in all examined banana tissues but at lower level in peel than in pulp. According to tissues, MA-EIL genes were differentially regulated by ripening and ethylene in mature green fruit and wounding in old and young leaves. MA-EIL2/AB266318 was the unique ripening- and ethylene-induced gene; MA-EIL1, MA-EIL4/Ab266320 and AB266321 genes were downregulated, while MA-EIL3/AB266319 presented an unusual pattern of expression. Interestingly, a marked change was observed mainly in MA-EIL1 and MA-EIL3/Ab266319 mRNA accumulation concomitantly with changes in ethylene responsiveness of fruit. Upon wounding, the main effect was observed in MA-EIL4/AB266320 and AB266321 mRNA levels, which presented a markedly increase in both young and old leaves, respectively. Data presented in this study suggest the importance of a transcriptionally step control in the regulation of EIL genes during banana fruit ripening.

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry (Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO4, NO3, NH4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d(-1) based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest Bat the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Effects of alkalis, oxidants and urea on the nutritive value of rhodes grass (Chloris gayana cv. Callide)

Two in sacco experiments were conducted to evaluate the impact on the nutritive value of rhodes grass hay (Chloris gayana cv. Callide) of treatment with alkalis or oxidants. In Experiment 1, three alkalis (Ca(OH)(2), NaOH, CaO) and two oxidants (NaOCl and H2O2) were applied at levels of 0, 20, 40, 60 or 80 g/kg of dry matter (DM). NaOH, Ca(OH)(2) and CaO had negative linear effects (P < 0.05) on the neutral detergent fibre (NDF) content and positive linear effects (P < 0.05) on the 48 h in sacco disappearances of DM, organic matter (OM), NDF and acid detergent fibre (ADF). NaOCl reduced (P < 0.05) NDF content but had no effect (P > 0.05) on the in sacco disappearances. H2O2 had no effect (P > 0.05) on the composition or digestibility of rhodes grass hay. In Experiment 2, effects of urea (0, 20, 40, 60 and 80 g urea/kg DM) and water (250, 500 and 750 g/kg DM) treatment of rhodes grass hay were examined in a 5 x 3 factorial experiment. Significant interactions between water and urea (P < 0.05) occurred for concentrations of crude protein (CP) and NDF, and 48 h in sacco disappearances of DM, OM (OMD) and NDE The combinations of water (g/kg DM) and urea (g/kg DM) that resulted in the highest concentrations of CP (281 g/kg DM) and OMD (747 g/kg DM) were 250 + 80 and 500 + 80, respectively. NaOH, Ca(OH)(2), CaO and urea significantly alter the NDF content and digestibility of rhodes grass hay, and urea also increases its CP content. Overall, NaOH was the most efficacious, followed by Ca(OH)(2), CaO, urea, NaOCl and H2O2. Crown Copyright (C) 2002 Published by Elsevier Science B.V. All rights reserved.

Ranolazine Exerts Potent Effects on Atrial Electrical Properties and Abbreviates Atrial Fibrillation Duration in the Intact Porcine Heart

Introduction: In vitro studies and ambulatory ECG recordings from the MERLIN TIMI-36 clinical trial suggest that the novel antianginal agent ranolazine may have the potential to suppress atrial arrhythmias. However, there are no reports of effects of ranolazine on atrial electrophysiologic properties in large intact animals. Methods and Results: In 12 closed-chest anesthetized pigs, effects of intravenous ranolazine (similar to 9 mu M plasma concentration) on multisite atrial effective refractory period (ERP), conduction time (CT), and duration and inducibility of atrial fibrillation (AF) initiated by intrapericardial acetylcholine were investigated. Ranolazine increased ERP by a median of 45 ms (interquartile range 29-50 ms; P < 0.05, n = 6) in right and left atria compared to control at pacing cycle length (PCL) of 400 ms. However, ERP increased by only 28 (24-34) ms in right ventricle (P < 0.01, n = 6). Ranolazine increased atrial CT from 89 (71-109) ms to 98 (86-121) ms (P = 0.04, n = 6) at PCL of 400 ms. Ranolazine decreased AF duration from 894 (811-1220) seconds to 621 (549-761) seconds (P = 0.03, n = 6). AF was reinducible in 1 of 6 animals after termination with ranolazine compared with all 6 animals during control period (P = 0.07). Dominant frequency (DF) of AF was reduced by ranolazine in left atrium from 11.7 (10.7-20.5) Hz to 7.6 (2.9-8.8) Hz (P = 0.02, n = 6). Conclusions: Ranolazine, at therapeutic doses, increased atrial ERP to greater extent than ventricular ERP and prolonged atrial CT in a frequency-dependent manner in the porcine heart. AF duration and DF were also reduced by ranolazine. Potential role of ranolazine in AF management merits further investigation. (J Cardiovasc Electrophysiol, Vol. 20, pp. 796-802, July 2009).

Population pharmacokinetics of perhexiline from very sparse, routine monitoring data

Using NONMEM, the population pharmacokinetics of perhexiline were studied in 88 patients (34 F, 54 M) who were being treated for refractory angina. Their mean +/- SD (range) age was 75 +/- 9.9 years (46-92), and the length of perhexiline treatment was 56 +/- 77 weeks (0.3-416). The sampling time after a dose was 14.1 +/- 21.4 hours (0.5-200), and the perhexiline plasma concentrations were 0.39 +/- 0.32 mg/L (0.03-1.56). A one-compartment model with first-order absorption was fitted to the data using the first-order (FO) approximation. The best model contained 2 subpopulations (obtained via the $MIXTURE subroutine) of 77 subjects (subgroup A) and 11 subjects (subgroup B) that had typical values for clearance (CL/F) of 21.8 L/h and 2.06 L/h, respectively. The volumes of distribution (V/F) were 1470 L and 260 L, respectively, which suggested a reduction in presystemic metabolism in subgroup B. The interindividual variability (CV%) was modeled logarithmically and for CL/F ranged from 69.1% (subgroup A) to 86.3% (subgroup B). The interindividual variability in V/F was 111%. The residual variability unexplained by the population model was 28.2%. These results confirm and extend the existing pharmacokinetic data on perhexiline, especially the bimodal distribution of CL/F manifested via an inherited deficiency in hepatic and extrahepatic CYP2D6 activity.

Interaction of temperature and vegetative flush maturity influences shoot structure and development of lychee (Litchi chinensis Sonn.)

Potted lychee trees (cv. Tai so) of varying vegetative flush maturity were grown under a range of temperature regimes and monitored for subsequent shoot structure and development. A combination of low temperature (15/17 or 18/13 degreesC day/night) and high vegetative flush maturity was necessary for floral initiation to occur, Exposure to high temperatures (28/23 degreesC) invariably resulted in the production of vegetative shoots, irrespective of flush maturity. Strong floral initiation was marked by the emergence of terminal particles and accompanying axillary particles. A decrea,;e in vegetative flush maturity or increase in temperature (e.g. 23/18 degreesC) resulted in a decrease in axillary shoot formation and the production of several intermediate shoot structures. These included leafy particles, stunted particles, partially emerged buds and non-emergent swollen buds, often produced on the same tree. At 23/18 degreesC, closer synchronisation of initial flush maturity was required for the production of a consistent shoot-type. Trees with synchronised mature flushes (I-2) at 23/18 degreesC resulted in the production of swollen terminal buds. Healthy trees were maintained in this state for at least 11 months. These results indicate that both temperature and flush maturity can influence subsequent shoot structure of lychee. In the absence of either a strong floral temperature (18/13 degreesC) or strong vegetative temperature (28/23 degreesC), slight differences in initial flush maturity have gteater impact on the type of emerging shoot formed. (C) 2002 Elsevier Science B.V. All rights reserved.

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.