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A novel graphene oxide (GO)-based carrier was fabricated for the controlled release of Benazepril (BENA). Freeze dried samples of GO–BENA carrier were prepared for controlled drug release at different pHs (pH = 2, 7, and 10) and release kinetics indicate BENA desorption from GO is by Fickian diffusion. The BENA yield from the carrier amounted to ~55% of the adsorbed material in a strongly acidic medium after 50 h. Binding fractions of BENA to 10 mg/L GO was determined for different solution concentrations of the drug. In vitro assays of cell proliferation (WST-1 kit), cell structural integrity (LDH kit) and flow cytometric indicators of necrosis in three different cell lines (CACO-2, SGC-7901, and primary mouse hepatic fibroblast) all demonstrated that the GO carrier had a good biocompatibility. The pH-dependent release sensitivity of the GO-based carrier suggests that it is a potential candidate for use in the controlled release of drugs in the acidic environment of the stomach.

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.

The spider Ornithoctonus huwena is a venomous spider found in southern China. Its venom is a complex mixture of numerous biologically active components. In this study, 41 novel unique transcripts encoding cellular proteins or other possible venom components were generated from the previously constructed cDNA library. These proteins were also annotated by KOG (eukaryotic orthologous group) and GO (gene ontology) terms. A novel cellular transcript contig encoding an EF-hand protein (named HWEFHP1) was found, which might be involved in the secretion of toxins in the venom glands. In order to have an overview of the molecular diversity of the O. huwena venom, the datasets of all the transcripts, peptides and proteins known so far were analyzed. A comparison of the data obtained through a proteomic versus a transcriptomic approach, revealed that only 15 putative cystine knot toxins (CKTs) were identified by both approaches, 29 transcripts coding for CKTs were found in the transcriptome but not as translated peptides in the venom proteome. However, no cellular protein with identical molecular weight was identified by both approaches. Our data may contribute to a deeper understanding of the biology and ecology of O. huwena and the relationship between structure and function of individual toxins.

Jingzhaotoxin-34 (JZTX-34) is a 35-residue polypeptide from the venom of Chinese tarantula Chilobrachys jingzhao . Our previous work reported its full-length cDNA sequence encoding a precursor with 87 residues. In this study we report the protein expression and biological function characterization. The toxin was efficiently expressed by the secretary pathway in yeast. Under whole-cell patch-clamp mode, the expressed JZTX-34 was able to inhibit tetrodotoxin-sensitive (TTX-S) sodium currents (IC 50 ∼ 85 nM) while having no significant effects on tetrodotoxin-resistant (TTX-R) sodium currents on rat dorsal root ganglion neurons. The inhibition of TTX-S sodium channels was completely reversed by strong depolarization (+120 mV). Toxin treatment altered neither channel activation and inactivation kinetics nor recovery rate from inactivation. However, it is interesting to note that in contrast to huwentoxin-IV, a recently identified receptor site-4 toxin from Ornithoctonus huwena venom, 100 nM JZTX-34 caused a negative shift of steady-state inactivation curve of TTX-S sodium channels by approximately 10 mV. The results indicated that JZTX-34 might inhibit mammalian sensory neuronal sodium channels through a mechanism similar to HWTX-IV by trapping the IIS4 voltage sensor in the resting conformation, but their binding sites should not overlay completely.

By sequencing random clones from the venom gland cDNA library of the spider Ornithoctonus huwena, a transcript, named SHL-Ib1, encoding a lectin-like peptide was cloned. The amino acid sequence of the putative mature peptide of SHL-Ib1 is identical, except for seven different residues, with that of SHL-I, a lectin found in the venom of O. huwena. The mature peptides of SHL-Ib1b and SHL-Ib1c are the mutants of SHL-Ib1 with two or three amino acid residues truncated at the C-terminal. The recombinant SHL-Ib1b and SHL-Ib1c were expressed successfully by the yeast expression system and purified by using a combination of ion-exchange and reverse phase high performance liquid chromatography (HPLC). The molecular masses of the two expressed peptides were identified by mass spectrometry, indicating that the C-terminals of the two peptides were not amidated. The two peptides can agglutinate human erythrocytes at minimal concentrations of 0.75 and 1.475mg/ml, respectively. Structure modeling of SHL-Ib1 has given a clue to the low agglutination bioactivities of these recombinant toxins. These lectin-like peptides, due to the small molecular sizes, may have the advantage to investigate the binding mechanism of the lectin and have the potential to be the carrier for drug delivery.

Formation of the gastrula organizer requires suppression of ventralizing signals and, in fish and frog, the need to counteract the effect of ubiquitously present maternal factors that activate the expression of Bmps. How the balance between dorsalizing and ventralizing factors is shifted towards organizer establishment at late blastula stages is not well understood. Mutations in zebrafish bozozok (boz) cause severe defects in axial mesoderm and anterior neurectoderm and affect organizer formation. The boz gene encodes the homeodomain protein Bozozok/Dharma and its expression in the region of the organizer is activated through β-catenin signaling. Here, we investigate the molecular mechanism by which boz contributes to the establishment of the organizer. We demonstrate that the homeodomain protein Boz acts as a transcriptional repressor in zebrafish: overexpression of an En-Boz fusion protein can rescue the boz phenotype, whereas a VP16-Boz fusion protein acts as an antimorph. Expression analysis of bmp2b indicates that Boz negatively regulates bmp2b in the prospective organizer. We demonstrate that this Boz activity is independent of that of other zygotic genes, because it also occurs when translation of zygotic genes is suppressed by cycloheximide(CHX). We identify two high-affinity binding sites for Boz within the first intron of the bmp2b gene. Deletion of these control elements abolishes Boz-dependent repression of bmp2b in the early blastula. Thus, Boz directly represses bmp2b by binding to control elements in the bmp2b locus. We propose that early transcriptional repression of bmp2b by Boz is one of the first steps toward formation of a stable organizer, whereas the later-acting Bmp antagonists (e.g. Chordin, Noggin)modulate Bmp activity in the gastrula to induce patterning along the dorsoventral axis. Thus, similar to Drosophila Dpp, asymmetry of Bmp expression in zebrafish is initiated at the transcriptional level, and the shape of the gradient and its function as a morphogen are later modulated by post-transcriptional mechanisms.

The Drosophila morphogenetic protein Bicoid (Bcd) is a homeodomain-containing activator that stimulates the expression of target genes during early embryonic development. We demonstrate that a small domain of Bcd located immediately N-terminally of the homeodomain represses its own activity in Drosophila cells. This domain, referred to as a self-inhibitory domain, works as an independent module that does not rely on any other sequences of Bcd and can repress the activity of heterologous activators. We further show that this domain of Bcd does not affect its properties of DNA binding or subcellular distribution. A Bcd derivative with point mutations in the self-inhibitory domain severely affects pattern formation and target gene expression in Drosophila embryos. We also provide evidence to suggest that the action of the self-inhibitory domain requires a Drosophila co-factor(s), other than CtBP or dSAP18. Our results suggest that proper action of Bcd as a transcriptional activator and molecular morphogen during embryonic development is dependent on the downregulation of its own activity through an interaction with a novel co-repressor(s) or complex(es).

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spider Selenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact and in situ S-carboxymethylated toxin. In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys2-Cys17, Cys9-Cys22, and Cys16-Cys29.