Determination of CO₂ sensitivity of micro-organisms in shaken bioreactors. II. Novel online monitoring method

In the present study, a new online monitoring method for the determination of the CO? sensitivity of micro-organisms, based on the values of the respiration factors [OTR (oxygen transfer rate) and CTR (carbon dioxide transfer rate)], obtained by using the RAMOS (respiratory activity monitoring system) device considering a variety of aeration rates in the measuring flask, is investigated. Based on the data of the OTR, obtained by RAMOS under a variety of specific aeration rates, the proposed new method was developed as an online monitoring method for CO? sensitivity of micro-organisms in shaken bioreactors. A maximum accumulated CO? concentration of 12% was derived in applied methods, provided that the cultivation system is carried out under optimal conditions. Additionally, to predict these conditions, an unsteady-state gas transfer model in shaken bioreactors would be very advantageous. The data of OTR obtained using the RAMOS device were analysed and recalculated by a programme considering the calibration factor (Cf). The major advantage of the new method is the possibility to determine the metabolic activity, regardless of manual sampling.     

One‐step separation and purification of rupestonic acid and chrysosptertin B from Artemisia rupestris L. by high‐speed counter‐current chromatography

Introduction – Artemisia rupestris L. is a well‐known traditional Chinese medicinal plant in Xinjiang. Rupestonic acid is the main active ingredient of A. rupestris L., and has been chosen as a ‘marker compound’ for the chemical evaluation or quality control of A. rupestris L. and its products. Although HSCCC separation method was developed before, the separation was performed with two steps using the same solvent system, which were time‐consuming and waste of the solvents. Objective – To develop a simple HSCCC method for the separation and purification of rupestonic acid in a single run. Methodology – The measurement of partition coefficient (K) was introduced to select the two‐phase solvent system. The simple HSCCC method was established according to the selected solvent system for separation and purification of rupestonic acid. The purity of target compound was test by HPLC and the structure was identified by MS, ¹H NMR and ¹³C NMR. Results – A total of 72.3 mg of rupestonic acid and 53.5 mg of chrysosptertin B with over 95% purity were yielded from 500 mg extracts of Artemisia rupestris L. in one‐step separation. Conclusion – The rupestonic acid was separated in a single run by HSCCC. Copyright © 2009 John Wiley & Sons, Ltd.     

Population genetic evidence for the east–west division of the narrow-barred Spanish mackerel (<Emphasis Type="Italic">Scomberomorus commerson,</Emphasis> Perciformes: Teleostei) along Wallace’s Line

Mitochondrial DNA D-loop (control) region (426-bp) was used to infer the genetic structure of Spanish mackerel (Scomberomorus commerson) from populations in Southeast Asia (Brunei, East and West Malaysia, Philippines, Thailand, Singapore, and China) and northern Australia (including western Timor). An east–west division along Wallace’s Line was strongly supported by a significant AMOVA, with 43% of the total sequence variation partitioned among groups of populations. Phylogenetic and network analyses supported two clades: clade A and clade B. Members of clade A were found in Southeast Asia and northern Australia, but not in locations to the west (Gulf of Thailand) or north (China). Clade B was found exclusively in Southeast Asia. Genetic division along Wallace’s Line suggests that co-management of S. commerson populations for future sustainability may not be necessary between Southeast Asian nations and Australia, however all countries should share the task of management of the species in Southeast Asia equally. More detailed genetic studies of S. commerson populations in the region are warranted.     

Characeae-derived carbonate deposits in Lake Ganau, Kurdistan Region, Iraq

Characeae, a family of calcifying green algae, are common in carbonate-rich freshwaters. The southwestern shoreline of Lake Ganau (Kurdistan Region, northeastern Iraq) harbors dense and thick mats of these algae (genus Chara). On the lake bottom and along the shore, carbonate sands and rocks rich in the remains of stems, branches, nodes, and whorls of Chara are deposited. These deposits show all stages of growth and degradation of characean algae, including replacement and lithification into limestone. The replacement of the fragments by fine-grained calcite preserved delicate microstructures of Chara, such as cortical walls, cell shape, inner and outer layers of the stems, and reproductive organs. Based on roundness, sorting, the degree of lithification, and preserved microstructures of the grains (fragments), three facies were recognized. The first is represented by a newly formed lime sand facies showing elongated grains, poor sorting, and reduced roundness, with pristine preservation of characean surface microstructures. The second is a weathered lime sand facies, which shows better sorting and good roundness, whereas internal structures of characean fragments are still well preserved. The third is comprised of a lithified lime sand facies (grainstone), with very well sorted and rounded grains, and poorly preserved external and internal structures of the characeans. As compared to the newly formed lime sand facies, the grainstone facies shows an increase in grain size by more than 30 %, owing to precipitation of micritic lamina of possible microbial origin. Eventually, the Characeae-derived lime sands are lithified into oolitic limestones with sparry calcite cement, forming a grainstone microfacies. The present study has important implications for the interpretation of pre-Quaternary environments, as it records all stages of the fossilization process of characean green algae and highlights the role of these algae in the formation of oolitic carbonate rocks.     

Conservation genetics and phylogeography of the poorly known Middle Eastern terrapin Mauremys caspica (Testudines: Geoemydidae)

The West Asian stripe-necked terrapin Mauremys caspica is widespread throughout the Middle East—a region for which only few phylogeographic studies are available. Due to landscape alteration, pollution and intensification of water management, M. caspica is increasingly threatened. However, genetic diversity among and within populations is poorly known, impeding the identification of management units. Using a nearly rangewide sampling, we analyzed 14 microsatellite loci and mtDNA sequences in order to gain insight into the population structure and history of M. caspica. In agreement with a previous study, we found two clusters of mitochondrial haplotypes, with one cluster distributed in the east and the other in the west of the range. However, our microsatellite data suggested a more pronounced geographical structuring. When null alleles were coded as recessive with structure 2.3.2, three clusters were revealed, with one cluster matching roughly the range of the western mitochondrial cluster, and the composite ranges of the two other microsatellite clusters correspond to the distribution of the eastern mitochondrial cluster. Naïve structure analyses without correction for null alleles were congruent with respect to the two eastern microsatellite clusters, but subdivided the western cluster into two units, with an additional geographical divide corresponding to the ‘Anatolian diagonal’—a well-known high mountain barrier impeding exchange between western and eastern taxa. In naïve analyses, the westernmost microsatellite cluster (from Central Anatolia) is quite isolated from the others, and its distinctness is also supported by fixation indices resembling the values among the other three clusters. One of the two eastern clusters is distributed in the Caucasus region plus Iran, and terrapins from Saudi Arabia and Bahrain constitute the second eastern cluster, supporting the view that these endangered populations are native. Coalescent-based analyses of our microsatellite data reveal for all four clusters bottlenecks 4,000–20,000 years ago, suggesting that climatic fluctuations of the Late Pleistocene and Holocene played an important role in shaping current genetic diversity. We propose that each of the four identified clusters, including the Central Anatolian one, should be treated as a distinct management unit. The presence of non-native terrapins in the animal trade of Bahrain highlights the danger of genetic pollution of the endangered Arabian populations. Further sampling is needed to elucidate the situation in southern and central Iran and Iraq. Our results confirm that genetic data do not support the validity of any of the three morphologically defined subspecies of M. caspica, and we propose that their usage be abandoned.     

Chemometric-assisted kinetic-spectrophotometric method for simultaneous determination of ascorbic acid, uric acid, and dopamine

A chemometric-assisted kinetic spectrophotometric method has been developed for simultaneous determination of ascorbic acid (AA), uric acid (UA), and dopamine (DA). This method relies on the difference in the kinetic rates of the reactions of analytes with a common oxidizing agent, tris(1,10-phenanthroline) and iron(III) complex (ferritin, [Fe(phen)₃]³⁺) at pH 4.4. The changes in absorbance were monitored spectrophotometrically. The data obtained from the experiments were processed by chemometric methods of artificial neural network (ANN) and partial least squares (PLS). Acceptable techniques of prediction set, randomization t test, cross-validation, and Y randomization were applied for the selection of the best chemometric method. The results showed that feedforward artificial neural network (FFANN) is more efficient than the other chemometric methods. The parameters affecting the experimental conditions were optimized, and it was found that under optimal conditions Beer’s law is followed in the concentration ranges of 4.3–74.1, 4.3–78.3, and 2.0–33.0 μM for AA, UA, and DA, respectively. The proposed method was successfully applied to the determination of analytes in serum and urine samples.     

LC/MS evaluation of metabolism and membrane transport of bombesin peptides

Two bombsin peptides, GRPR agonist [Aca-QWAVGHLM-NH₂] and antagonist [fQWAVGHL-NHEthyl] were evaluated. We employed the highly sensitive Waters Q-Tof Premier MS coupled with a UPLC system to identify the metabolites produced by rat hepatocytes or PC-3 human prostate cancer cells; and we utilized the AB/MDS 4000 Q-Trap LC/MS/MS system with highly sensitive quantitative and qualitative performance, to quantitatively analyze the internalization of GRPR agonist and antagonist in PC-3 cells. The major metabolites of both GRPR agonist and antagonist were the result of peptide bond hydrolysis between W and A which was demonstrated by observation of the N-terminal fragment m/z 446 (Aca-QW-OH) for agonist and m/z 480 (fQW-OH) for antagonist. Both peptides were also hydrolyzed between A and V which formed peaks m/z 517 [Aca-QWA-OH] and m/z 555 (VGHLM-NH2) for the agonist and m/z 551 [fQWA-OH] and m/z 452 (VGHL-NHEthyl) for the antagonist. The peptide agonist also formed a unique metabolite that resulted from hydrolysis of the C-terminal amide. The antagonist showed significantly slower metabolism as compared to the agonist in both rat hepatocytes and PC-3 cells. The antagonist also showed significantly lower PC-3 cell internalization rate than that of the agonist. In conclusion, the metabolism profiles of both GRPR agonist and antagonist peptides were identified by LC/MS. The antagonist peptide was more stable than the agonist peptide in rat hepatocyte incubation. One major factor could be the hydrolysis-resistant C-terminal L-NHEthyl group compared with the unsubstituted amide of the agonist. Another factor could be different amino acid sequences of the agonist and antagonist that may also influence the enzymatic hydrolysis. The antagonist ligand is potentially more useful for receptor-targeted imaging due primarily to its higher metabolic stability.     

Synthesis and Characterization of Novel Chickpea Protein Hydrolysate-Vanadium Complexes Having Cell Inhibitory Effects on Lung Cancer A549 Cells Lines

The Protein Journal - Designing new types of drugs with preferred properties against cancer is a great issue for scientists dealing with synthesis and study of biological activity. Several...     

αTubulin 67C and Ncd Are Essential for Establishing a Cortical Microtubular Network and Formation of the Bicoid mRNA Gradient in Drosophila

The Bicoid (Bcd) protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localized at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfil the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. We report hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (i) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (ii) long MTs up to 50 µm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the bcd mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. Our data strongly corroborate the ARTS model and explain the occurrence of the bcd mRNA gradient.