Assessment of Microzooplankton Grazing on <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis> Using a Species- Specific Approach Combining Quantitative Real-Time PCR (QPCR) and Dilution Methods

Delaware’s Inland Bays (DIB) are subject to numerous mixed blooms of harmful raphidophytes each year, and Heterosigma akashiwo is one of the consistently occurring species. Often, Chattonella subsalsa, C. cf. verruculosa, and Fibrocapsa japonica co-occur with H. akashiwo, indicating a dynamic consortium of raphidophyte species. In this study, microzooplankton grazing pressure was assessed as a top–down control mechanism on H. akashiwo populations in mixed communities. Quantitative real-time polymerase chain reaction (QPCR) with species-specific primers and probes were used in conjunction with the dilution method to assess grazing pressure on H. akashiwo and other raphidophytes. As a comparison, we measured changes in chlorophyll a (chl a) to determine whole community growth and mortality caused by grazing. We detected grazing on H. akashiwo using QPCR in samples where chl a analyses indicated little or no grazing on the total phytoplankton community. Overall, specific microzooplankton grazing pressure on H. akashiwo ranged from 0.88 to 1.88 day⁻¹ at various sites. Experiments conducted on larger sympatric raphidophytes (C. subsalsa, C. cf. verruculosa and F. japonica) demonstrated no significant microzooplankton grazing on these species. Grazing pressure on H. akashiwo may provide a competitive advantage to other raphidophytes such as Chattonella spp. that are too large to be consumed at high rates by microzooplankton and help to shape the dynamics of this harmful algal bloom consortium. Our results show that QPCR can be used in conjunction with the dilution method for evaluation of microzooplankton grazing pressure on specific phytoplankton species within a mixed community. An erratum to this article can be found at     

Cytosolic and non-cytosolic carbonic anhydrase enzymes from bovine leukocytes.

In this study, carbonic anhydrase (CA) enzyme has been purified and separately characterized according to bound form in 4 steps as outer peripheral, cytosolic, inner peripheral, and integral from bovine leukocyte. Affinity chromatography has also been used for purification of the enzyme in four steps. CA has been found for each step. Measurment of enzyme activity has been done by CO2 hydratase activity and esterase activity methods. Optimum pH and optimum temperature have been defined for each step of purified enzyme. The behaviors of CA with specific inhibitors, such as KSCN and NaN3 have been investigated. In each step, molecular weight and purity have been determined by gel filtration and SDS-PAGE electrophoresis. In addition, enzyme K(M) and Vmax values have been determined with the method of Lineweaver-Burk.     

Some metals inhibit the glutathione S‐transferase from Van Lake fish gills

Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play important role cellular signaling. The present article focuses on the role of Cd²⁺, Cu²⁺, Zn²⁺, and Ag⁺ in vitro inhibition of GST. For this purpose, GST was purified from Van Lake fish (Chalcalburnus tarichii Pallas) gills with 110.664 EU mg^?1 specific activity and 79.6% yield using GSH‐agarose affinity chromatographic method. The metal ions were tested at various concentrations on in vitro GST activity. IC₅₀ values were found for Cd⁺², Cu⁺², Zn⁺², Ag⁺ as 450.32, 320.25, 1510.13, and 16.43 μM, respectively. K _i constants were calculated as 197.05 ± 105.23, 333.10 ± 152.76, 1670.21 ± 665.43, and 0.433 ± 0.251 μM, respectively. Ag⁺ showed better inhibitory effect compared with the other metal ions. The inhibition mechanisms of Cd²⁺ and Cu²⁺ were non‐competitive, whereas Zn²⁺ and Ag⁺ were competitive. Co²⁺, Cr²⁺, Pb²⁺, and Fe³⁺ had no inhibitory activity on GST.     

Effects of putrescine and ethephon on some oxidative stress enzyme activities and proline content in salt stressed spinach leaves

The effects of putrescine and ethephon on peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.14.18.1), catalase (CAT; EC 1.11.1.6) activities and proline content in spinach leaves under saline stress were investigated. In control conditions, putrescine increased PPO and CAT activities and proline content, but decreased POD activity. Ethephon increased these three enzyme activities but did not affect proline content. In saline conditions, putrescine increased POD and CAT activities and proline content, while it decreased PPO activity. Ethephon increased both PPO and CAT activities and proline content, but decreased POD activity. Putrescine and ethephon have opposite effects on the enzyme activities and proline accumulation because they acts as antagonists.     

Glucose transporter expression in rat embryo and uterus during decidualization, implantation, and early postimplantation.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

Lipid peroxidation and antioxidant enzyme activities in erythrocytes of type I and II alcoholics

Reduced and oxidized glutathione (GSH and GSSG), protein-bound glutathione, lipid peroxidation and antioxidant enzyme activities were determined in the erythrocyte lysates and membranes of type I and II alcoholics in order to clarify the effect of age-of-onset and the duration of the alcohol consumption on erythrocyte oxidant and antioxidant status. The osmotic fragility and susceptibility of the erythrocytes to haemolysis were also determined. Erythrocyte lipid peroxidation was significantly increased but, GSH and protein-bound GSH, GSH/GSSG ratio and antioxidant enzyme activities were markedly decreased in the erythrocytes of the alcoholic subgroups. Erythrocyte count and haemoglobin content in the blood of alcoholics were found to be decreased in accordance with the finding that erythrocytes were more fragile and less resistant to haemolysis particularly in type II alcoholics. The present study showed that ethanol-induced oxidative stress in erythrocytes can lead to haemolysis and membrane-specific injuries in erythrocytes of the alcoholic subtypes.     

Apoptosis contributes to vascular lumen formation and vascular branching in human placental vasculogenesis

Placental vasculogenesis consists of several stages, including appearance of hemangioblasts and angiogenic cell islands, setting up a primitive vascular network, and transition from vasculogenesis to sprouting and nonsprouting angiogenesis. In the present study, we hypothesized that placental vasculogenesis and angiogenesis require apoptosis during the formation of primitive vascular pattern, vessel elongation, and angiogenic branching. Vasculogenesis and apoptotic cells were identified using CD31 immunohistochemistry, hematoxylin-eosin (H-E) staining, CD31-TUNEL double-labeling, and transmission-electron microscopy (TEM). No TUNEL-positive cell was detected in angiogenic cell islands; however, several TUNEL-positive cells were observed during the primitive lumen formation. Interestingly, some of the stromal cells located between vasculogenic areas during the endothelial tube elongation and angiogenic branching also were TUNEL-positive. The presence of morphological aspects of apoptosis, such as nuclear shrinkage and nuclear bodies (apoptotic bodies), also was confirmed in H-E-stained and TEM-depicted sections. Quantitative analysis showed that higher ratios for apoptotic cells were found in the core stroma of villi among the vascular branching areas and in the primitive capillary lumen compared to angiogenic cell cords and vasculatures with advanced lumens (P < 0.05). In conclusion, our results suggest that apoptosis likely is involved in the physiologic mechanisms of placental vasculogenesis and angiogenesis, such as lumen formation and angiogenic branching.     

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid‐based nanofiber membrane

In this study, polyacrylic acid‐based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid‐based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid‐based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Ca²⁺ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis–Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis–Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE‐based biosensors.