The 1.2 A resolution crystal structure of TcpG, the Vibrio cholerae DsbA disulfide-forming protein required for pilus and cholera-toxin production

The enzyme TcpG is a periplasmic protein produced by the Gram-negative pathogen Vibrio cholerae. TcpG is essential for the production of ToxR-regulated proteins, including virulence-factor pilus proteins and cholera toxin, and is therefore a target for the development of a new class of anti-virulence drugs. Here, the 1.2 Å resolution crystal structure of TcpG is reported using a cryocooled crystal. This structure is compared with a previous crystal structure determined at 2.1 Å resolution from data measured at room temperature. The new crystal structure is the first DsbA crystal structure to be solved at a sufficiently high resolution to allow the inclusion of refined H atoms in the model. The redox properties of TcpG are also reported, allowing comparison of its oxidoreductase activity with those of other DSB proteins. One of the defining features of the Escherichia coli DsbA enzyme is its destabilizing disulfide, and this is also present in TcpG. The data presented here provide new insights into the structure and redox properties of this enzyme, showing that the binding mode identified between E. coli DsbB and DsbA is likely to be conserved in TcpG and that the [beta]5-[alpha]7 loop near the proposed DsbB binding site is flexible, and suggesting that the tense oxidized conformation of TcpG may be the consequence of a short contact at the active site that is induced by disulfide formation and is relieved by reduction

Auditory Deficit as a Consequence Rather than Endophenotype of Specific Language Impairment: Electrophysiological Evidence

Are developmental language disorders caused by poor auditory discrimination? This is a popular theory, but behavioural evidence has been inconclusive. Here we studied children with specific language impairment, measuring the brain's electrophysiological response to sounds in a passive paradigm. We focused on the T-complex, an event-related peak that has different origins and developmental course from the well-known vertex response.We analysed auditory event-related potentials to tones and syllables from 16 children and 16 adolescents with specific language impairment who were compared with 32 typically-developing controls, matched for gender, IQ and age.We replicated prior findings of significant reduction in Ta amplitude for both children and adolescents with specific language impairment, which was particularly marked for syllables. The topography of the T-complex to syllables indicated a less focal response in those with language impairments. To distinguish causal models, we considered correlations between size of the Ta response and measures of language and literacy in parents as well as children. The best-fitting model was one in which auditory deficit was a consequence rather than a cause of difficulties in phonological processing.The T-complex to syllables has abnormal size and topography in children with specific language impairment, but this is more likely to be a consequence rather than a cause of difficulties in phonological processing

Protein disulphide isomerase-assisted functionalization of proteinaceous substrates

Protein disulphide isomerase (PDI) is an enzyme that catalyzes thiol-disulphide exchange reactions among a broad spectrum of substrates, including proteins and low-molecular thiols and disulphides. As the first protein-folding catalyst reported, the study of PDI has mainly involved the correct folding of several cysteine-containing proteins. Its application on the functionalization of protein-based materials has not been extensively reported. Herein, we review the applications of PDI on the modification of proteinaceous substrates and discuss its future potential. The mechanism involved in PDI functionalization of fibrous protein substrates is discussed in detail. These approaches allow innovative applications in textile dyeing and finishing, medical textiles, controlled drug delivery systems and hair or skin care products.We thank to FCT 'Fundacao para a Ciencia e Tecnologia' (scholarship SFRH/BD/38363/2007) for providing Margarida Fernandes the grant for PhD studies

The multidrug resistance IncA/C transferable plasmid encodes a novel domain swapped dimeric protein disulfide isomerase

Background: Bacterial IncA/C plasmids distribute antibiotic resistance genes and encode a conserved thioredoxin-fold protein (DsbP). Results: DsbP shuffles incorrect disulfide bonds in misfolded proteins, and its structure diverges from previously characterized disulfide isomerases. Conclusion: Plasmid-encoded DsbP is a novel domain-swapped protein-disulfide isomerase. Significance: IncA/C plasmids may encode this protein proofreading machinery to ensure horizontal gene transfer of antibiotic resistance genes

Growth Hormone Responses to Thyroid Hormone in the Neonatal Rat: RESISTANCE AND ANAMNESTIC RESPONSE

Differences in the growth hormone (GH) responses to primary and to secondary stimulation with triiodothyronine (T3) were studied in rats deprived of thyroid hormone from birth. Neonatal hypothyroidism was induced in pups by feeding pregnant rats an iodine-deficient, propylthiouracil-containing diet. T3 stimulation was carried out in pups by subcutaneous injection of a single dose of 50 μg T3/100 g body wt. Pituitary GH content, rate of GH synthesis in vitro, and GH messenger (m)RNA activity in a cellfree translation system were measured. No significant differences in body weight and in pituitary GH content were observed between hypothyroid and normal pups at ages 1, 3, and 6 d. 10- and 28-d-old hypothyroid pups showed a significant arrest of growth, decreased pituitary GH content, and development of GH responsiveness to T3. In contrast, serum thyroxine concentration in hypothyroid pups was <0.15 μg/dl, significantly lower than normal at all ages. GH synthesis and GH mRNA activity studied in pituitaries of 28-d-old rats were expressed as percent total protein synthesis and percent mRNA activity, respectively. GH synthesis and mRNA activity were 3.0 and 2.6% in hypothyroid rats, 3.3 and 2.9% in hypothyroid rats given a single T3 injection 14 d earlier (T3-withdrawn rats), and 26.8 and 27.1% in normal rats. Administration of T3 to hypothyroid rats induced an increase in GH synthesis and GH mRNA activity, reaching 5.8 and 5.6% 12 h after primary stimulation and 12.2 and 16.1% 12 h after secondary stimulation. The response rates were linear but 2.5-fold more rapid after secondary stimulation. The latter response was similar to that observed after T3 stimulation of rats rendered hypothyroid during adulthood. The responses of GH synthesis and mRNA activity were concordant after both primary and secondary T3 stimulation. A twofold increase in both parameters was observed as early as 2 h after T3 injection. Four conclusions can be drawn from these experiments. First, during neonatal life, GH accumulation in rat pituitaries is independent of thyroid hormone and is insensitive to T3. Second, GH dependence on and sensitivity to thyroid hormone is acquired between the 6th and 10th d of neonatal life. Third, secondary T3 stimulation produces an anamnestic response manifested by an increased rate of GH synthesis and mRNA activity. Fourth, primary T3 stimulation is not associated with a lag in the endogenous translation of the newly accumulated GH mRNA