AMPA receptor expression in mouse testis and spermatogonial GC-1 cells: A study on its regulation by excitatory amino acids

Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d -aspartate ( d -Asp) and N-methyl- d -aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC-1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC-1 cells. Subsequently, GC-1 cells were incubated with medium containing l -Glu, d -Glu, d -Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA-treated GC-1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p-extracellular signal-regulated kinase (ERK), p-Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l -Glu-, d -Glu-, and NMDA-treated GC-1 cells. At 30 minutes and 2 hours of incubation, treated GC-1 cells showed significantly higher expression levels of p-ERK and p-Akt. A consequent increase of PCNA and Aurora B expressions was induced by l -Glu and NMDA, but not by d -Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA-treated GC-1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.     

The role of chemical fingerprinting: application to Ephedra

Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in Traditional Chinese Medicine (TCM). Preparations, namely teas, of E. sinica have been used for over 5000 years as a stimulant and as an antiasthmatic. In the West, extracts of E. sinica, E. intermedia or E. equisetina are most commonly used in dietary supplements as a stimulant and to promote weight loss. More than 50 species of Ephedra are native to both hemispheres, but the detection of ephedrine alkaloids has been limited to species in Eurasia. Currently, methods exist to quantitate the ephedrine alkaloids in extracts of plant material or dietary supplements, but the methods are not able to verify the extract is of an Ephedra species. Reverse phase high performance liquid chromatography with photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two regions of comparison were determined in the chromatograms at 320 nm. The series of peaks between 52 and 64 min confirms an Ephedra species is being analyzed. The aforementioned peaks also could distinguish between Ephedra species from Eurasia, North America and South America. Peaks at ca. 57 and 59 min were isolated and determined to be two new compounds, 4-(2-eicosyloxycarbonyl-vinyl)-benzoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid respectively. Authentication of ground plant material as Ephedra can be achieved by this chemical fingerprinting method.     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.     

Snowflake Vitreoretinal Degeneration (SVD) Mutation R162W Provides New Insights into Kir7.1 Ion Channel Structure and Function

Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of I_Kir7.1 and positive shift in ‘0’ current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.     

Novel Large Apolipoprotein E-Containing Lipoproteins of Density 1.006–1.060 g/ml in Human Cerebrospinal Fluid

Abstract: Although the critical role of apolipoprotein E (apoE) allelic variation in Alzheimer's disease and in the outcome of CNS injury is now recognized, the functions of apoE in the CNS remain obscure, particularly with regard to lipid metabolism. We used density gradient ultracentrifugation to identify apoE-containing lipoproteins in human CSF. CSF apoE lipoproteins, previously identified only in the 1.063–1.21 g/ml density range, were also demonstrated in the 1.006–1.060 g/ml density range. Plasma lipoproteins in this density range include low-density lipoprotein and high-density lipoprotein (HDL) subfraction 1 (HDL₁). The novel CSF apoE lipoproteins are designated HDL₁. No immunoreactive apolipoprotein A-I (apo A-I) or B could be identified in the CSF HDL₁ fractions. Large lipoproteins 18.3 ± 6.6 nm in diameter (mean ± SD) in the HDL₁ density range were demonstrated by electron microscopy. Following fast protein liquid chromatography of CSF at physiologic ionic strength, apoE was demonstrated in particles of average size greater than particles containing apoA-I. The largest lipoproteins separated by this technique contained apoE without apoA-I. Thus, the presence of large apoE-containing lipoproteins was confirmed without ultracentrifugation. Interconversion between the more abundant smaller apoE-HDL subfractions 2 and 3 and the novel larger apoE-HDL₁ is postulated to mediate a role in cholesterol redistribution in brain.     

Isolation and Characterisation of Insulin-Releasing Compounds from <Emphasis Type="Italic">Pseudechis australis</Emphasis> and <Emphasis Type="Italic">Pseudechis butleri</Emphasis> Venom

Crude venom from two elapid snakes Pseudechis australis and Pseudechis butleri was fractionated by gel filtration chromatography and selected fractions screened for in vitro insulin-releasing activity using clonal pancreatic BRIN-BD11 cells. Following acute 20-min incubation at 5.6 mM glucose, 9 fractions exhibited significant (P < 0.001) insulin-releasing activity. Structural characterisation of active fractions was achieved primarily using MALDI–TOF MS and N-terminal Edman degradation sequencing. The partial N-terminal sequences are reported for a total of 7 venom components. Their homology to existing sequences as determined using BLAST searching uncovered the main insulin-releasing families as being phospholipases A₂ and short α-neurotoxins. A number of sequences are reported for the first time from P. butleri venom which is much less studied than the related P. australis.