The Ni(II) complex of the tripeptide NH 2 -glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc) 2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGHecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 Å of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.

A mollusk-specific toxin, TxVIIA, having potent paralytic activity was isolated from the venom of sea snail Conus textile (Fainzilber M et al., 1991, Eur JBiochem 202:589-595). The structure reported above was based upon amino acid analysis and the Edman degradation. We have recently reinvestigated this toxin employing some of the most novel techniques in mass spectrometry. We now report a revised structure based primarily on high-energy collision-induced dissociation analysis of the two Asp"-N peptides of the reduced, pyridinylethyl derivative representing the entire sequence using matrix-assisted laser desorption ionization (MALDI) as CGGYSTYCyVDSy CCSDNCVRSYCTLF-NH2 (y, y-carboxyglutamic acid or Gla).The N-terminus of the previous sequence was incorrect, apparently due to a side reaction of reduction and alkylation, which led to the erroneous assignment of Trp for the N-terminal residue.In addition, the last two C-terminal amino acids and the C-terminal amidation had not been detected. Also, a combination of electrospray ionization mass spectrometry and positive and negative ion MALDI mass spectrometry provided information on the molecular weights of the native and derivatized toxin and presence of two Gla residues. Thus, TxVIIA does not have an "unusual" sequence as previously reported, but in fact belongs to the conserved Cys framework for W -and 6-conotoxins. However, the four net negative charges with the cysteine-rich structure of this revised sequence is highly unusual for conopeptides.

BackgroundAcute mastitis is a common disease occurring in postpartum lactating women. This study aimed to identify the high-risk factors for suppurative mastitis (SM) in women who were breastfeeding.Material/MethodsA total of 262 non-SM patients and 63 SM patients admitted to the Department of Breast and Thyroid Surgery of Liaocheng People’s Hospital were retrospectively enrolled in this study. The high-risk factors for SM during lactation were determined by assessing admission time after acute mastitis, galactostasis, primipara or pluripara, nipple injuries, antibiotic treatment course, and diabetes.ResultsThe incidence of SM in patients with admission time greater than 72 h after occurrence of acute mastitis was significantly higher than that in patients whose admission time was less than 72 h (p<0.0001). Patients with galactostasis were more prone to develop SM compared to patients without galactostasis (p<0.0001), and the incidence of SM in primiparous patients was significantly higher than that in pluriparous patients (p=0.003). The incidence rate of SM was significantly higher in patients with diabetes than in patients without diabetes (p=0.002). Patients with nipple injuries were more prone to suffer from SM relative to the patients without nipple injuries (p<0.0001). However, there was no significant difference in the incidence of SM according to the antibiotic treatment course (p=0.134).ConclusionsSM during lactation was closely associated with admission time after the occurrence of acute mastitis, galactostasis, primipara, diabetes, and nipple injuries.

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