Synthesis of Disulfide Surrogate Peptides Incorporating Large-Span Surrogate Bridges Through a Native-Chemical-Ligation-Assisted Diaminodiacid Strategy.

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide-containing peptides. However, peptides incorporating large-span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)-assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible-to-obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X-ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.

Chemical synthesis of membrane proteins by the removable backbone modification method.

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Robust Chemical Synthesis of Membrane Proteins through a General Method of Removable Backbone Modification.

Chemical protein synthesis can provide access to proteins with post-translational modifications or site-specific labelings. Although this technology is finding increasing applications in the studies of water-soluble globular proteins, chemical synthesis of membrane proteins remains elusive. In this report, a general and robust removable backbone modification (RBM) method is developed for the chemical synthesis of membrane proteins. This method uses an activated O-to-N acyl transfer auxiliary to install in the Fmoc solid-phase peptide synthesis process a RBM group with switchable reactivity toward trifluoroacetic acid. The method can be applied to versatile membrane proteins because the RBM group can be placed at any primary amino acid. With RBM, the membrane proteins and their segments behave almost as if they were water-soluble peptides and can be easily handled in the process of ligation, purification, and mass characterizations. After the full-length protein is assembled, the RBM group can be readily removed by trifluoroacetic acid. The efficiency and usefulness of the new method has been demonstrated by the successful synthesis of a two-transmembrane-domain protein (HCV p7 ion channel) with site-specific isotopic labeling and a four-transmembrane-domain protein (multidrug resistance transporter EmrE). This method enables practical synthesis of small- to medium-sized membrane proteins or membrane protein domains for biochemical and biophysical studies.

Peptide toxins and small-molecule blockers of BK channels.

Large conductance, Ca(2+)-activated potassium (BK) channels play important roles in the regulation of neuronal excitability and the control of smooth muscle contractions. BK channels can be activated by changes in both the membrane potential and intracellular Ca(2+) concentrations. Here, we provide an overview of the structural and pharmacological properties of BK channel blockers. First, the properties of different venom peptide toxins from scorpions and snakes are described, with a focus on their characteristic structural motifs, including their disulfide bond formation pattern, the binding interface between the toxin and BK channel, and the functional consequence of the blockage of BK channels by these toxins. Then, some representative non-peptide blockers of BK channels are also described, including their molecular formula and pharmacological effects on BK channels. The detailed categorization and descriptions of these BK channel blockers will provide mechanistic insights into the blockade of BK channels. The structures of peptide toxins and non-peptide compounds could provide templates for the design of new channel blockers, and facilitate the optimization of lead compounds for further therapeutic applications in neurological disorders or cardiovascular diseases.

[Study on epidemiological effect of the freeze-dried attenuated live varicella vaccine].

OBJECTIVE: To evaluate the safety and epidemiological effect of the Freeze-dried Live attenuated varicella vaccine. METHODS: A random, double-blind control clinical trial was adopted. RESULTS: In the observation period, the incidence of varicella was 0.8 per thousand in the experimental group and 8.7 per thousand in control group. There was a significant difference (B.P=0.000017). Vaccine effectiveness (VE(%)) was 90.8%, the lower limit of 95%CI was 88.7%. CONCLUSION: The varicella vaccine produced by Changchun keygen biological products co., Ltd. was safe and effective.

Glycine uptake regulates hippocampal network activity via glycine receptor-mediated tonic inhibition.

Functional glycine receptors (GlyRs) are enriched in the hippocampus, but their role in hippocampal function remains unclear. Since the concentration of ambient glycine is determined by the presence of powerful glycine transporter (GlyT), we blocked the reuptake of glycine in hippocampal slices to examine the role of GlyRs. Antagonists of GlyT type 1 (GlyT1) but not that of GlyT type 2 (GlyT2) induced excitatory postsynaptic potential (EPSP)-spike depression, which was reversed by the specific GlyR antagonist strychnine. Moreover, endogenously elevating the glycine concentration with the GlyT1 antagonists facilitated NMDA receptor-dependent long-term potentiation induction, and elicited a strychnine-sensitive chloride current. In addition, impairment of glial function with fluoroacetate blocked the effect of GlyT1 antagonists on the EPSP-spike curve. Furthermore, pretreatment with sarcosine was effective in controlling pentylenetetrazol-induced seizures. These results indicate an essential role of GlyTs in fine-tuning tonic activation of GlyRs and suggest a potential role of GlyR-dependent EPSP-spike depression in hippocampal network stability.

Glycine receptor activation regulates short-term plasticity in CA1 area of hippocampal slices of rats.

Functional glycine receptors (GlyRs) are enriched in the hippocampus, but their roles in synaptic transmission are unclear. In this study, we examined the effect of GlyR activation on paired-pulse stimulation of the whole-cell postsynaptic currents (PSCs) in the Schaffer-CA1 synapses in rat hippocampal slices. Bath application of glycine reduced the amplitude of PSCs, accompanied by an increase in holding current and resting conductance. Moreover, glycine application increased the paired-pulse ratio (PPR) of PSCs significantly, an effect largely abolished by the GlyR specific antagonist strychnine. Interestingly, glycine application had no significant effect on either the amplitude or the PPR of excitatory postsynaptic currents (EPSCs). Our findings suggest that GlyR activation regulates hippocampal short-term plasticity by altering GABAergic neurotransmission.