MicroRNA-197 controls ADAM10 expression to mediate MeCP2's role in the differentiation of neuronal progenitors.

Duplication of MECP2 (Methyl-CpG-binding protein 2) causes severe mental illness called MECP2 duplication syndrome (MDS), yet the underlying mechanism remains elusive. Here we show, in Tg(MECP2) transgenic mouse brain or cultured neural progenitor cells (NPCs), that elevated MeCP2 expression promotes NPC differentiation into neurons. Ectopic expression of MeCP2 inhibits ADAM10 and thus the NOTCH pathway during NPC differentiation. In human cells, this downregulation on ADAM10 was mediated by miRNA-197, which is upregulated by MeCP2. Surprisingly, miR-197 binds to the ADAM10 3'-UTR via its 3' side, not the canonical seed sequence on the 5' side. In mouse cells, a noncoding RNA Gm28836 is used to replace the function of miR-197 between MeCP2 and ADAM10. Similar to MeCP2, overexpressing miR-197 also promotes NPCs differentiation into neurons. Interestingly, three rare missense mutations (H371R, E394K, and G428S) in MECP2, which we identified in a Han Chinese autism spectrum disorders (ASD) cohort showed loss-of-function effects in NPC differentiation assay. These mutations cannot upregulate miR-197. Overexpressing miR-197 together with these MeCP2 mutations could rescue the downregulation on ADAM10. Not only the inhibitor of miR-197 could reverse the effect of overexpressed MeCP2 on NPCs differentiation, but also overexpression of miR-197 could reverse the NPCs differentiation defects caused by MECP2 mutations. Our results revealed that a regulatory axis involving MeCP2, miR-197, ADAM10, and NOTCH signaling is critical for NPC differentiation, which is affected by both MeCP2 duplication and mutation.

Tracing the evolution of venom phospholipases A2 in Gloydius strauchii and related pitvipers: A tale of two acidic isozymes.

Two acidic Asp49-PLA2s with Glu6 substitution and a neutral Lys49-PLA (designated Gst-K49) were cloned from G. strauchii venom glands, their full amino acid sequences were deduced. The predominant acidic PLA2 (designated Gst-E6a) contains 124 residues and the M18W30 substitutions, while the minor acidic PLA2 (designated Gst-E6b) contains 122 residues and the V18A30 substitutions. Their sequences are most similar to those of the respective orthologous PLA2s of G. intermedius venom. Gst-E6a and Gst-E6b appear to be paralogs and possibly have different predatory targets or functions. The LC-MS/MS results indicate the presence of only three PLA2 gene products in the crude venom, the relative expression levels were in the order of Gst-E6a â‰« Gst-E6b > Gst-K49, as confirmed by qPCR results. In contrast to other Gloydius, G. strauchii venom does not contain neurotoxic or basic anticoagulant Asp49-PLA2s, but Gst-K49 is the first Lys49-PLA2 identified in Gloydius venoms. However, its venom content is relatively low and its pI value 7.3 is much lower than those of other Lys49-PLA2s and. The Lys49-PLA2 genes appear to regress in the venom of most of Gloydius and related rattlesnake, and this evolutionary regression occurred before the dispersal of Asian pitvipers to the New World.

Serine protease isoforms in Gloydius intermedius venom: Full sequences, molecular phylogeny and evolutionary implications.

Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1-4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification. Specific amidolytic activities of the fractions from HPLC and anion exchange separation were assayed using four chromogenic substrates, respectively. Molecular phylogenetic tree based on the sequences of these vSPs and their orthologs revealed six major clusters, one of them covered four lineages of plasminogen activator like vSPs. N-glycosylation patterns and variations for the vSPs are discussed. The high sequence similarities between G. intermedius vSPs and their respective orthologs from American pitvipers suggest that most of the isoforms evolved before Asian pitvipers migrated to the New World. Our results also indicate that the neurotoxic venoms contain more kallikrein-like vSPs and hypotensive components than the hemorrhagic venoms. SIGNIFICANCE: Full sequences and expression levels of nine paralogous serine proteases (designated as GiSPs) of Gloydius intermedius venom have been studied. A kallikrein-like enzyme is most abundant and four isoforms homologous to venom plasminogen-activators are also expressed in this venom. Taken together, the present and previous data demonstrate that the neurotoxic G. intermedius venoms contain more hypotensive vSPs relative to other hemorrhagic pitviper venoms and the pitviper vSPs are highly versatile and diverse. Their structure-function relationships remain to be explored and compared. A novel, simplified phylogenetic tree based on the sequences of GiSPs and their closely related orthologs from other pitvipers reveals six major subtypes and offers a better understanding of vSP duplication and evolution in pitvipers of both the Old and New Worlds. It is well known that specific vSPs are potential therapeutic or diagnostic agents that target the plasma proteins or coagulation factors. Our results not only render deeper insights into the variation and evolution of vSPs, but may help to choose right venoms for the development of better therapeutic leads.

Transcriptome and proteome of the highly neurotoxic venom of Gloydius intermedius.

The venomics of Gloydius intermedius were investigated using expressed sequence tags (ESTs) analyses, 2D gel-electrophoresis combined with MALDI-TOF/TOF, and LC-MS/MS. A total of 1920 ESTs from the venom gland cDNA library were sequenced; 74% of them belonged to toxin-families. The four most abundant families among the toxin transcripts were: serine protease (SP, 36.2%), bradykinin potentiating peptide (25.3%), l-amino acid oxidase (LAAO, 13.1%), and phospholipase A2 (PLA2, 9.9%). Moreover, the full sequences of four PLA2s, eight SPs, cysteine-rich secretory protein (CRISP), C-type-lectin-like-protein (CTLP), hyaluronidase, metalloproteinase, and nerve growth factor were deduced from the cDNA sequences. Excluding the CRISP and hyaluronidase, most of the G. intermedius venom proteins bear 92-99% sequence identities to those of other pitviper venoms. The most abundant components are PLA2s (37%), SPs (20%) and LAAO (6%), while metalloproteinase, CTLP, and other components each account for <3% of the total venom proteins. The abundance of Gintexin (a crotoxin-like neurotoxin) and low levels of hemorrhagic metalloproteases, disintegrins and CTLPs highlight the great venom differences between G. intermedius and other hemorrhagic pitvipers. The bimorphism of hemorrhagic and neurotoxic venoms among Gloydius is confirmed; our results shed more lights on the co-evolution of both neurotoxicity and hypotension in some viperid venoms.

Structures and functions of crotoxin-like heterodimers and acidic phospholipases A2 from Gloydius intermedius venom: Insights into the origin of neurotoxic-type rattlesnakes.

The cDNAs encoding four major phospholipases A2 (PLA2s) were sequenced while the expressed sequence tags of Gloydius intermedius venom glands were constructed. These PLA2s were designated as Gintexin-A precursor, Gintexin-B, Gin-E6a and Gin-E6b, respectively. The deduced amino acid sequences of the former two PLA2s are 80% and 90% identical to those of crotoxin-A-precursor and crotoxin-B1, respectively. We also purified Gintexin-A, Gintexin-B, Gin-E6a and Gin-E6b like PLA2 from the venom. The latter three PLA2s are enzymatically active but not strongly anticoagulant for human plasma. Gin-E6a and E6b-like PLA2s induced mouse platelet aggregation but inhibited rabbit platelet aggregation. The isolated Gintexin, a 1:1 complex of Gintexin-A and Gintexin-B, blocked the twitch of chick biventer cervicis tissue presynaptically. Results of N-terminal sequencing and peptide mass fingerprinting reveal that Gintexin-A undergoes proteolytic processing similar to crotoxin-A. This is the first time heterodimeric ß-neurotoxins are found in Asian pitviper venom, and incompatible neurotoxic- and hemorrhagic-type venoms are found to evolve in parallel within the genus Gloydius, like in Crotalus. Thus, G. intermedius probably is the ancestor of rattlesnakes with type-II venom, and characterization of its venomics helps us to understand the evolution of heterodimeric neurotoxic PLA2s and the paedomorphic trend observed in Neotropical rattlesnake venoms. BIOLOGICAL SIGNIFICANCE: For the first time, a heterodimeric neurotoxic PLA2 (designated as Gintexin) has been isolated from the venom of an Asian pitviper, which shows a characteristic venom gland transcriptome similar to those of the neurotoxic type rattlesnakes. The fact that the venom of G. intermedius is less hemorrhagic than those of other Gloydius species, reveals that incompatible neurotoxic- and hemorrhagic-type venoms have evolved in parallel within the genus Gloydius, like the genus Crotalus. Our findings suggest that G. intermedius is the most probable ancestor of some Neotropical rattlesnakes. The results may revolutionize the theory regarding the origin of type-II rattlesnakes and assist with the diagnosis and clinical management of G. intermedius bites. Furthermore, the possibility of using the currently available antivenoms of Neotropical rattlesnakes to treat G. intermedius bites seems feasible.

Analysis of high-frequency stimulation-evoked synaptic plasticity in mouse hippocampal CA1 region.

Extracellular recordings of field excitatory postsynaptic potential (fEPSP) is one of the most common ways for studies of synaptic plasticity, such as long-term potentiation (LTP) and paired-pulse plasticity (PPP). The measurement of the changes in the different components of fEPSP waveform, such as the initial slope, initial area, peak amplitude and whole area, were commonly used as criteria for the judgement of potentiation or depression of synaptic plasticity. However, the differences in the conclusions drawn from measuring different components of fEPSP waveform at the same recording have still been largely ignored. Here we compared high-frequency stimulation (HFS)-evoked synaptic plasticity, both LTP and PPP, by measuring different components of fEPSP waveform, including the initial slope, initial area, peak amplitude, whole area and time course. The results not only indicated the acceleration of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor kinetics underlies LTP in hippocampal CA1 region of mice, but also showed that different measurements of fEPSP waveform at the same recording result in different magnitudes of LTP and different forms of PPP in hippocampal CA1 region of mice. After HFS, the paired-pulse ratio was slightly decreased by measurement of the initial area, but obviously increased by measurement of the initial slope of the pair fEPSPs. These results might draw apparently contradictory conclusions. Therefore, careful and complete analysis of the data from different parts of fEPSP waveforms is important for reflection of the faithful changes in synaptic plasticity.

Anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells provokes potent T-lymphocyte activation and cytotoxicity.

The targeting of tumor cells by cytotoxic T lymphocytes is a promising strategy for biotherapy, but T cells require 2 signals via the T-cell receptor - CD3 complex and CD28 molecules for activation. To bridge the gap between cytotoxic T lymphocytes and tumor cells, our objective in this study was to describe the construction and the cell surface-anchored expression of a fusion protein, anti-CD3 scFv-B7.1, derived from inserting a fusion gene encoding anti-CD3 scFv and the extra-cellular domain of B7.1 fused by the splicing by overlap extension method into a mammalian expression vector, pDisplay. Transfection of the recombinant vector by electroporation into HeLa cells resulted in the production of protein migrating at approximately 57 kDa under reducing conditions. The expressed fusion protein could bind to T lymphocytes and induce strong T-cell activation. Meanwhile, a potent cytotoxicity was induced in the mixed culture of T-cell-modified tumor cells in a 96 h methyl-thiazolyl-diphenyl tetrazolium bromide assay. Our results indicate that this bifunctional protein, through activating T lymphocytes to lyse homologous human carcinomas, may be of potential value for T-cell-based immunotherapeutical treatment protocols in vivo.

[Eukaryotic expression of anti-CD3 single chain Fv antibody gene and the characterization of its bioactivities].

AIM: To express anti-CD3 scFv in Hela cells and investigate its biological activity. METHODS: DNA fragment encoding anti-CD3 scFv was inserted into eukaryotic expression vector pDisplay. The recombinant expression vector was sequenced and then transfected into Hela cells by electroporation method. The expression of anti-CD3 scFv was identified by in situ hybridization. In-vitro T lymphocyte activation was then detected by (3)H-TdR incoporation method. Anti-CD3 scFv gene-transfected Hela cells were co-cultured with T cells and cytotoxicity was measured by MTT colorimetry. RESULTS: Anti-CD3 scFv gene was correctly inserted into pDisplay and expressed in Hela cells. The secreted anti-CD3 scFv was able to activate T lymphocytes in the presence of anti-CD28 mAb. Cytotoxicity could be observed when anti-CD3 scFv gene-transfected Hela cells were mixed and co-cultured with T lymphocytes. CONCLUSION: Anti-CD3 scFv expressed by Hela cells can activate T lymphocytes.