Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α–δ subunits. Three isoforms (αδ-BgTx-1–3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17–0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve–muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α–δ site as compared with α–ε or α–γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs

Protective Effects of Dimethyl Sulfoxide on Labile Protein Interactions during Electrospray Ionization

Electrospray ionization mass spectrometry is a valuable tool to probe noncovalent interactions. However, the integrity of the interactions in the gas-phase is heavily influenced by the ionization process. Investigating oligomerization and ligand binding of transthyretin (TTR) and the chaperone domain from prosurfactant protein C, we found that dimethyl sulfoxide (DMSO) can improve the stability of the noncovalent interactions during the electrospray process, both regarding ligand binding and the protein quaternary structure. Low amounts of DMSO can reduce in-source dissociation of native protein oligomers and their interactions with hydrophobic ligands, even under destabilizing conditions. We interpret the effects of DMSO as being derived from its enrichment in the electrospray droplets during evaporation. Protection of labile interactions can arise from the decrease in ion charges to reduce the contributions from Coulomb repulsions, as well as from the cooling effect of adduct dissociation. The protective effects of DMSO on labile protein interactions are an important property given its widespread use in protein analysis by electrospray ionization mass spectrometry (ESI-MS)

Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis.

Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant

Relative inductions of CAMP gene transcript by colostrum, transitional milk, mature milk and infant formulas.

<p>(<b>A</b>) HT-29 cells were stimulated for 48 h with 50 g/l hydrophilic fractions of breast milk from different lactation periods. The median of the relative induction of <i>CAMP</i> gene transcript was 2.5-fold for colostrum (1–3 days postpartum, pp), 2.1-fold for transitional milk (4–10 days pp) and 3.9-fold for mature milk (11- days pp) samples. The median of 50 g/l hydrophilic fraction of infant formulas was 2.2, but with a high variability in <i>CAMP</i> gene induction. (<b>B</b>) HT-29 cells were stimulated with the hydrophilic fraction of breast milk collected from one mother from day 7 to 19 pp. A positive linear correlation was observed between <i>CAMP</i> gene transcript induction and time pp (R² = 0.5728, p-value 0.043). (<b>A and B</b>) Each sample is performed in triplicates.</p

Isolation of the CAMP gene inducing component.

<p>(<b>A</b>) Heat treatment (100°C) of the hydrophilic fraction of breast milk for 30 min did not affect <i>CAMP</i> gene inducing capacity in HT-29 cells after 48 h of stimulation. Breast milk components were separated into high and low molecular weight components (more or less than 10 kDa) and the <i>CAMP</i> gene inducing capacity of breast milk was retained only in the low molecular weight fraction (≤10 kDa). Displays the mean and SD of at least three independent experiments in triplicate. (<b>B</b>) The low molecular fraction was subjected to cationic exchange and the chromatographic fractions were used for stimulation of HT-29 cells for 48 h. Material in fraction 29, eluted at 4% buffer B, resulted in a 10-fold induction of <i>CAMP</i> gene transcript. (<b>C</b>) Material in fraction 29, from (<b>B</b>), was separated by size exclusion chromatography and obtained fractions were assayed for <i>CAMP</i> gene transcript in HT-29 cells after 48 h of stimulation. A 6.5-fold induction of <i>CAMP</i> gene was observed by stimulation with material from fraction 42. (<b>B–C</b>) The X-axes denote the elution volume. The grey bars representing the activity are shown as a mean of the fold induction performed in triplicate.</p

Induction of CAMP gene transcript in different cell lines stimulated with lactose.

<p>(<b>A</b>) HT-29 cells were stimulated with 10–70 g/l lactose for 48 h and <i>CAMP</i> gene transcript was monitored. At 20 g/l a 3.6-fold expression was observed that increased dose-dependently up to 11.3-fold at 70 g/l. (<b>B</b>) HT-29 cells were stimulated with 60 g/l lactose for 4, 24 and 48 h. After 24 h and 48 h an 8- and 17-fold induction of <i>CAMP</i> gene transcript was observed. No induction of <i>CAMP</i> gene was observed at 4 h. (<b>C</b>) T84 cells were stimulated with 60 g/l lactose for 4, 24 and 48 h. A 3.5-fold enhanced level of <i>CAMP</i> gene transcript was observed after 24 h and was 11.3-fold after 48 h. (<b>D</b>) THP-1 monocytes (black line) and differentiated macrophage-like THP-1 cells (grey line) were stimulated with 60 g/l lactose for 4, 24 and 48 h. In monocytes, 7.2 and 25.7-fold induction of <i>CAMP</i> gene transcript was detected after 24 h and 48 h, respectively. The macrophage-like cells exhibited a 13.5-fold induction of <i>CAMP</i> gene transcript after 24 h that declined to 5.6-fold after 48 h. (<b>A–D</b>) Displays the mean and SD of five independent experiments in duplicate.</p