Astrocyte–Endotheliocyte Axis in the Regulation of the Blood–Brain Barrier

Neurochemical Research - The evolution of blood–brain barrier paralleled centralisation of the nervous system: emergence of neuronal masses required control over composition of the...     

A Simple Method for the Preparation of D-α-Aminoadipic Acid

Abstract

High quantity (1 g and more) of racemically and chromatograph-ically pure D-α-aminoadipic acid was Drepared by selective metabolism of the L-isomer of the commercially available DL-α-aminoadi-pate by Pseudomonas putida. The overall yield of this preparation averaged 40%. The final product has a [a]²⁵ _D value of ?25°. This procedure can be useful in the synthesis of high purity D-α-amino-adipate, a compound shown recently to be a useful tool in the study of neurotransmission mechanism mediating synaptic excitation.     

Simple assay for adsorption of proteins to the air–water interface

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air–water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.     

Effect of Cyanotoxins on the Hypothalamic–Pituitary–Gonadal Axis in Male Adult Mouse

Background

Microcystins LR (MC-LR) are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic–Pituitary–Gonadal Axis (HPG) is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis.

Methods

Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day) for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH) and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro. Results: MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice.

Conclusions

MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice.     

Ranger Based Monitoring in the Virunga–Bwindi Region of East-Central Africa: A Simple Data Collection Tool for Park Management

Effective management of protected areas is dependent on information on the illegal and legal use of the habitat by people, the ecological and behavioural needs of key species, and trends in resource availability and ecological processes. The International Gorilla Conservation Programme working with the protected area authorities in Rwanda, Uganda and the Democratic Republic of Congo has developed a ranger based monitoring system, using basic protocols for data collection that guide protected area staff in park management. This programme is a key management tool for the three park authorities responsible for the conservation of the mountain gorilla (Gorilla beringei beringei) in the Virunga and Bwindi forest blocks. The programme was initiated in 1997 and has enabled the gathering of extensive information on illegal activities, key species of fauna and flora, and habituated and unhabituated groups of gorillas. Ranger based monitoring is a simple and cost effective tool that can be sustained in the parks in the Virunga–Bwindi region with limited external support. It provides park managers with information that prompts appropriate responses to threats to the ecosystem. For example information on the distribution of illegal activities determines targeted patrol coverage to address specific threats. The bottom-up approach of ranger based monitoring includes a strong capacity building component and empowers field staff in park management activities.     

Utilization of Methyl Proton Resonances in Cross-Saturation Measurement for Determining the Interfaces of Large Protein–Protein Complexes

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220–223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of ¹H₂O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Effect of Prenatal Stress on the Pituitary–Adrenal Axis in Blue Foxes

Handling is a source of stress for farm bred blue foxes. The influence of handling during the late gestation period on the pituitary–adrenal axis was studied in 10-day old male and female blue foxes. Cortisol and progesterone were measured by radioimmunoassay in the plasma, adrenal homogenates, and in vitro incubates from animals of both sexes. Adrenals were incubated in vitro in the absence or presence of ACTH. In addition, the adrenal weight and plasma concentration of ACTH were assessed. In cubs of both sexes, the adrenal weight was decreased after prenatal stress treatment. The plasma concentration of progesterone and the adrenal cortisol in vitro production were elevated in the prenatally stressed female cubs, as compared to the control, along with the adrenal progesterone in vitro production in prenatally stressed male cubs. The adrenal cortisol and progesterone content and plasma ACTH and cortisol concentrations were not affected by prenatal stress. In conclusion, the results of this study suggest that the prenatal stress induced by handling pregnant vixens can affect the pituitary–adrenal axis in neonatal offspring, this effect being more pronounced in female cubs.     

Studies on the mechanism of estrogen biosynthesis in the rat ovary–I

Methods were evaluated for obtaining a reliable, active estrogen synthetase (aromatase) system from the rat ovary for mechanistic studies. Short terrn treatment with luteinizing hormone and follicle stimulating hormone in various combinations did not produce appreciable stimulation, whereas long term treatment (8–16 days) with pregnant mare's serum gonadotropin increased activity in homogenates up to nine fold per mg wet wt of tissue. A similar increase per mg protein was noted in the 105,000_g microsomal fraction where the bulk of the activity was found. Various conditions for preparing and incubating the aromatase were evaluated to obtain optimal enzyme activity. The potencies of six steroids as aromatase inhibitors were compared in the rat ovarian and human placental microsomal systems. In all cases except one the results were comparable.     

A Novel Two-Stage Approach for Epistasis Detection in Genome-Wide Case–Control Studies

A significant challenge in epistasis detection is the huge amount of data, which leads to combinatorial explosion. This study focuses on a two-stage approach for detecting epistasis only among single nucleotide polymorphisms (SNPs) that show some marginal effect. We present this two-stage approach based on the fusion of two criteria (TwoFC) to detect epistatic interactions. We fuse the G ² test and absolute probability difference function as a scoring function to measure the strength of association between SNPs and disease status. The fused scoring function is an excellent measure of the strength of such an association. The two-stage strategy greatly reduces the computation load on epistasis detection. We use both simulated data sets and a real disease data set to evaluate our method. The results of an experiment on the simulated data sets show that TwoFC exhibits high power and sample efficiency. The results of an experiment on the real disease data set show that our method performs well even with large-scale data sets.     

The Gut–Brain Axis and Peroxisome Proliferator-Activated Receptors in the Regulation of Epileptogenesis

Journal of Evolutionary Biochemistry and Physiology - A large proportion of patients with epilepsy suffer from pharmacoresistant forms of the disease, and this makes the search for new treatments...     

Studies of the effect of environmental factors on the rotifer predator–prey system in freshwater

The aim of this work is to evaluate the effect of environmental factors: temperature and photoperiod on the zooplankton predator–prey system. Rotifers, an important and cosmopolitan group of zooplankton in freshwater, were used in our study. We investigated the effect of temperature (20, 23, and 30°C) and of photoperiod (L:D = 12:0 and 0:12) on the predatory rotifer Asplanchna brightwelli consuming rotifer Brachionus calyciflorus as prey. Under A. brightwelli predation, populations of B. calyciflorus prey were consumed more slowly at 20 ± 1 and 30 ± 1°C as compared to 23 ± 1°C. Prey consumption by A. brightwelli increased from 0.63 ± 0.09 ind. predator⁻¹ at 20°C to a peak of 1.22 ± 0.12 ind. predator⁻¹ at 23°C, then decreased significantly to 0.93 ± 0.14 ind. predator⁻¹ at 30 ± 1°C. In addition, predation responded to temperature changing sensitively and rapidly. Statistical analysis showed that the prey consumption were significant different under altered temperature periods during 12 h. Photoperiod also significantly influenced the rate of A. brighwelli predation. B. calyciflorus suffered less predation in darkness than in light. The rate of prey consumption in light (1.06 ind. predator⁻¹) was twice the average of that in darkness (0.51 ind. predator⁻¹). Furthermore, predation rate varied under changing photoperiod but predators moved back into the light did not resume their original consumption rate. Our results demonstrate that whether the predation in rotifer successfully or not is strongly influenced by temperature and photoperiod.     

Characterization of a Single-Chain Variable Fragment Recognizing a Linear Epitope of Aβ: A Biotechnical Tool for Studies on Alzheimer’s Disease?

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with devastating effects. Currently, therapeutic options are limited to symptomatic treatment. For more than a decade, research focused on immunotherapy for the causal treatment of AD. However, clinical trials with active immunization using Aβ encountered severe complications, for example meningoencephalitis. Consequently, attention focused on passive immunization using antibodies. As an alternative to large immunoglobulins (IgGs), Aβ binding single-chain variable fragments (scFvs) were used for diagnostic and therapeutic research approaches. scFvs can be expressed in E. coli and may provide improved pharmacokinetic properties like increased blood-brain barrier permeability or reduced side-effects in vivo. In this study, we constructed an scFv from an Aβ binding IgG, designated IC16, which binds the N-terminal region of Aβ (Aβ(1-8)). scFv-IC16 was expressed in E. coli, purified and characterized with respect to its interaction with different Aβ species and its influence on Aβ fibril formation. We were able to show that scFv-IC16 strongly influenced the aggregation behavior of Aβ and could be applied as an Aβ detection probe for plaque staining in the brains of transgenic AD model mice. The results indicate potential for therapy and diagnosis of AD.     

Contacts in Death: The Role of the ER–Mitochondria Axis in Acetic Acid-Induced Apoptosis in Yeast

Endoplasmic reticulum–mitochondria contact sites have been a subject of increasing scientific interest since the discovery that these structures are disrupted in several pathologies. Due to the emerging data that correlate endoplasmic reticulum–mitochondria contact sites function with known events of the apoptotic program, we aimed to dissect this interplay using our well-established model of acetic acid-induced apoptosis in Saccharomyces cerevisiae. Until recently, the only known tethering complex between ER and mitochondria in this organism was the ER–mitochondria encounter structure (ERMES). Following our results from a screening designed to identify genes whose deletion rendered cells with an altered sensitivity to acetic acid, we hypothesized that the ERMES complex could be involved in cell death mediated by this stressor. Herein we demonstrate that single ablation of the ERMES components Mdm10p, Mdm12p and Mdm34p increases the resistance of S. cerevisiae to acetic acid-induced apoptosis, which is associated with a prominent delay in the appearance of several apoptotic markers. Moreover, abrogation of Mdm10p or Mdm34p abolished cytochrome c release from mitochondria. Since these two proteins are embedded in the mitochondrial outer membrane, we propose that the ERMES complex plays a part in cytochrome c release, a key event of the apoptotic cascade. In all, these findings will aid in targeted therapies for diseases where apoptosis is disrupted, as well as assist in the development of acetic acid-resistant strains for industrial processes.     

A model for the rate of enzymatic hydrolysis of cellulose in heterogeneous solid–liquid systems

Hydrolysis of cellulose by cellulase enzymes has been studied in a stirred batch reactor at 50°C. A kinetic model has been devised by which the behaviour of such a reaction could be described. The model has been developed on the basis of shrinking particle theory and Langmuir isotherm concept. The applicability of the model has been tested by comparing the experimental results for diverse reaction systems, obtained in the present study or taken from the literature, and those predicted from the model. The degree of agreement was within ±2–11%.     

Development of a Highly Sensitive Immuno-PCR Assay for the Measurement of α-Galactosidase A Protein Levels in Serum and Plasma

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.     

Patterns of Stove Use in the Context of Fuel–Device Stacking: Rationale and Implications

EcoHealth - The implementation of clean fuel and stove programs that achieve sustained use and tangible health, environmental, and social benefits to the target populations remains a key challenge....     

Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.