The relationship between vascular endothelial growth factor and spermatogenesis disturbance in an experimentally-induced unilateral cryptorchidism murine model

Molecular Biology Reports - This study is to explore the relationship between vascular endothelial growth factor (VEGF) and pathological changes in cryptorchidism by using murine model of...     

Functional divergence analysis of vertebrate neuronal nicotinic acetylcholine receptor subunits

Nicotinic acetylcholine receptors (nAChRs) are pentamers formed by subunits from a large multigene family and are highly variable in kinetic, electrophysiological and pharmacological properties. Due to the essential roles of nAChRs in many physiological procedures and diversity in function, identifying the function-related sites specific to each subunit is not only necessary to understand the properties of the receptors but also useful to design potential therapeutic compounds that target these macromolecules for treating a series of central neuronal disorders. By conducting a detailed function divergence analysis on nine neuronal nAChR subunits from representative vertebrate species, we revealed the existence of significant functional variation between most subunit pairs. Specifically, 44 unique residues were identified for the α7 subunit, while another 22 residues that were likely responsible for the specific features of other subunits were detected. By mapping these sites onto the 3?D structure of the human α7 subunit, a structure-function relationship profile was revealed. Our results suggested that the functional divergence related sites clustered in the ligand binding domain, the β2–β3 linker close to the N-terminal α-helix, the intracellular linkers between transmembrane domains, and the “transition zone” may have experienced altered evolutionary rates. The former two regions may be potential binding sites for the α7* subtype-specific allosteric modulators, while the latter region is likely to be subtype-specific allosteric modulations of the heteropentameric descendants such as the α4β2* nAChRs.

Communicated by Ramaswamy H. Sarma     

Covalently linking the Escherichia coli global anaerobic regulator FNR in tandem allows it to function as an oxygen stable dimer

Glutathione (GSH) serves as an important anti-oxidant in the brain by scavenging harmful reactive oxygen species that are generated during different molecular processes. The GSH level in the brain provides indirect information on oxidative stress of the brain. We report in vivo detection of GSH non-invasively from various brain regions (frontal cortex, parietal cortex, hippocampus and cerebellum) in bilateral hemispheres of healthy male and female subjects and from bi-lateral frontal cortices in patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). All AD patients who participated in this study were on medication with cholinesterase inhibitors. Healthy young male (age 26.4±3.0) and healthy young female (age 23.6±2.1) subjects have higher amount of GSH in the parietal cortical region and a specific GSH distribution pattern (parietal cortex>frontal cortex>hippocampus ~ cerebellum) has been found. Overall mean GSH content is higher in healthy young female compared to healthy young male subjects and GSH is distributed differently in two hemispheres among male and female subjects. In both young female and male subjects, statistically significant (p=0.02 for young female and p=0.001 for young male) difference in mean GSH content is found when compared between left frontal cortex (LFC) and right frontal cortex (RFC). In healthy young female subjects, we report statistically significant positive correlation of GSH content between RFC and LFC (r=0.641, p=0.004) as well as right parietal cortex (RPC) and left parietal cortex (LPC) (r=0.797, p=0.000) regions. In healthy young male subjects, statistically significant positive correlation of GSH content was observed between LFC and LPC (r=0.481, p=0.032) regions. This statistical analysis implicates that in case of a high GSH content in LPC of a young male, his LFC region would also contain high GSH and vice versa. The difference in mean of GSH content between healthy young female control and female AD patients in RFC region (p=0.003) and difference in mean of GSH content between healthy young male control and male AD patients (p=0.05) in LFC region is found to be statistically significant. It is the first scientific report correlating alteration (in selective brain regions) of GSH level with clinical status of male and female subjects using non-invasive imaging technique.     

Up-regulation of Cavβ3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.     

Efficient expression and purification of recombinant alcohol oxidase in Pichia pastoris

In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD₆₀₀ 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H₂O₂ inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas.     

Biotransformation of phenylpyruvic acid to phenyllactic acid by growing and resting cells of a <Emphasis Type="Italic">Lactobacillus</Emphasis> sp.

Phenyllactic acid (PLA) is a novel antimicrobial compound derived from phenylalanine (Phe). Lactobacillus sp. SK007, having high PLA-producing ability, was isolated from Chinese traditional pickles. When 6.1 mM phenylpyruvic acid (PPA) was used to replace Phe as substrate at the same concentration, PLA production increased 14-fold and the fermentation time decreased from 72 h to 24 h with growing cells. With resting cells, however, 6.8 mM PLA could be obtained as optimal yield using the following conditions: 12 mM PPA, 55 mM glucose, pH 7.5, 35°C and 4 h.     

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (～60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.     

Discrete Notch signaling requirements in the specification of hematopoietic stem cells

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.