The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane ␤-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy-and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the ␤-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal ␤-barrel domain confirmed the feasibility of this mechanism.FepA is a Gram-negative bacterial outer membrane (OM) 6 protein that transports ferric enterobactin (FeEnt) (1-3). The crystal structures of FepA (4) and other bacterial metal transporters (FhuA, BtuB,57)), contain a C-terminal, 22-stranded ␤-barrel, placing them in the porin superfamily (5). Their ϳ150-residue globular N termini (N-domain; see Fig. 1) reside within their ␤-barrels. This architecture is potentially consistent with the "ball-and-chain" mechanism of membrane transport, whereby the globule controls solute (ligand) uptake by moving in and out of the channel. This process was postulated for nervous system channels (6), but no demonstrated examples of ball-and-chain transport are known.FepA and its relatives are unlike other porins (7, 8), because they selectively adsorb metal chelates with high affinity (3, 9 -14). Ligand binding causes small conformational changes that activate them to transport competency (15-17), hence their designation "ligand-gated porin" (LGP). The requirements for metabolic energy (18 -20) and another cell envelope protein, TonB (21-24), in LGP mediated transport are well known but unaccounted for: the OM has no source of energy and cannot sustain an ion gradient because of its open porin channels (7); TonB is a minor cell envelope protein whose functions are not yet understood.In live cells, FepA binds and transports FeEnt via sub-reactions with different dependences on energy and TonB. (i) In the absence of ligand the receptor opens, and its flexible surface loops extend outward (25). (ii) FeEnt binds to FepA in a biphasic reaction (26) that begins with adsorption to aromatic amino acids in the loop extremities (27, 28). Multiple determinants in multiple loops, including L7 (25), converge on the iron complex, creating a closed conformation that associates the negatively charged (Ϫ3), catecholate iron center with basic and aromatic residues in the r...

SummaryListeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10-100-fold lower affinity for ferric siderophores ( K m ª 1-10 nM) than Gramnegative bacteria, and generally lower uptake rates.

Escherichia coli

BackgroundThyroid nodules are common among adults though only a small percentage is malignant, which can histologically mimic benign nodules. Accurate diagnosis of these thyroid nodules is critical for the proper clinical management.MethodsWe investigated immunoexpression in 98 surgically removed benign thyroid nodules including 52 hyperplastic nodules (HN) and 46 follicular/Hurthle cell adenomas (FA), and 54 malignant tumors including 22 follicular carcinoma (FC), 20 classic papillary carcinoma (PTC), and 12 follicular variant papillary carcinoma (FVPC).ResultsThe staining results showed that malignant tumors express galectin-3, HBME-1, CK19 and Ret oncoprotein significantly more than benign nodules. The sensitivity of these markers for the distinction between benign and malignant lesions ranged from 83.3% to 87%. The sensitivity of two-marker panels was not significantly different. Immunoexpression was usually diffuse and strong in malignant tumors, and focal and weak in the benign lesions.ConclusionOur findings indicate that these immunomarkers are significantly more expressed in malignant tumors compared to benign lesions and may be of additional diagnostic value when combined with routine histology.

The results revealed that LncRNA GAS5 was upregulated while the miR-221 was downregulated in the tissues, plasma and exosomes of patients with CRC. The results of ROC showed that the expressions of both lncRNA GAS5 and miR-221 in the tissues, plasma and exosomes had diagnostic value in CRC. While the LncRNA GAS5 expression in tissues, plasma and exosomes were associated with the tumor node metastasis (TNM) stage, Dukes stage, lymph node metastasis (LNM), local recurrence rate and distant metastasis rate, the MiR-221 expression in tissues, plasma and exosomes were associated with tumor size, TNM stage, Dukes stage, LNM, local recurrence rate and distant metastasis rate. LncRNA GAS5 and miR-221 expression in tissues, plasma and exosomes were found to be independent prognostic factors for CRC. Following the overexpression of GAS5, the GAS5 expressions was up-regulated and miR-221 expression was down-regulated; the rate of cell proliferation, migration and invasion were decreased.

Colorectal cancer (CRC) is a frequently occurring lethal disorder with heterogeneous outcomes and drug responses. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) play a critical role in carcinogenesis. Hence, the aim of this study was to investigate the role of lncRNA growth arrest-specific 5 (GAS5) in CRC cells via mediation of the microRNA-222-3p (miR-222-3p)/GAS5/phosphatase and tensin homolog (PTEN)-signaling pathway. HCT116 and SW480 cells were collected and treated with small interfering (si)-lncRNA GAS5, overexpressing (oe)-lncRNA GAS5, miR-222-3p mimic, miR-222-3p inhibitor, or si-lncRNA GAS5 + miR-222-3p mimic. The miR-222-3p level and mRNA and protein levels of GAS5, Beclin1, light-chain 3B (LC3B), PTEN, and Akt were detected. Besides, cell migration, invasion, and apoptosis as well as acidic vesicular organelles (AVOs) were examined respectively. Xenografts in nude mice were also performed to detect tumorigenesis in vivo . Results suggested that the downregulation of lncRNA GAS5 decreased the expressions of Beclin1, LC3B, and PTEN. When treated with oe-lncRNA GAS5 or miR-222-3p inhibitor, HCT116 and SW480 cells exhibited suppressed invasion and migration abilities and increased apoptotic cells and autophagosome and AVO activities. Moreover, overexpression of GAS5 inhibited the tumorigenesis of CRC cells in vivo . Taken together, lncRNA GAS5 upregulated the expression of PTEN by functioning as a competing endogenous RNA (ceRNA) of miR-222-3p, thus inhibiting CRC cell migration and invasion and promoting cell autophagy.

Melanosis refers to abnormal or excessive deposition of melanin pigment in the cells and/or tissue, which can be seen in any organ but commonly in skin and oral mucosa. Melanosis of the urinary bladder is an extremely rare benign condition and only a handful of cases been reported in the English literature before. In this article, we report a new case of urinary melanosis, describe the differential diagnostic features from pseudomelanosis and offer clues for correct diagnosis. We also provide comprehensive review of the literature on the subject.

We have found that nanobacteria, recently discovered Gram-negative atypical bacteria, can cause local calciphylaxis on the mitral valve in a setting of high-calcium X phosphorous product in the blood. We present the case of a 33-year-old man with diabetic renal failure on continuous ambulatory peritoneal dialysis who died as a result of multiple brain infarcts due to embolizations from mitral valve vegetations. Systemic calciphylaxis was not present. Spectrometric analysis of the mitral valve vegetations showed that they were composed of calcium phosphate, carbonate apatite form, and fibrin. The electron microscopy of the thrombotic vegetation demonstrated nanobacterium as a nidus for carbonate apatite formation. Investigation for the presence of nanobacteria in the multiple organs involved in systemic calciphylaxis may be of help in elucidating the pathogenesis of this frequently fatal disorder.