The toxin HlyA is exported from Escherichia coli, without a periplasmic intermediate, by a type I system comprising an energized inner-membrane (IM) translocase of two proteins, HlyD and the traffic ATPase HlyB, and the outer-membrane (OM) porin-like TolC. These and the toxin substrate were expressed separately to reconstitute export and, via affinity tags on the IM proteins, cross-linked in vivo complexes were isolated before and after substrate engagement. HlyD and HlyB assembled a stable IM complex in the absence of TolC and substrate. Both engaged HlyA, inducing the IM complex to contact TolC, concomitant with conformational change in all three exporter components. The IM-OM bridge was formed primarily by HlyD, which assembled to stable IM trimers, corresponding to the OM trimers of TolC. The bridge was transient, components reverting to IM and OM states after translocation. Mutant HlyB that bound, but did not hydrolyse ATP, supported IM complex assembly, substrate recruitment and bridging, but HlyA stalled in the channel. A similar picture was evident when the HlyD C-terminus was masked. Export thus occurs via a contiguous channel which is formed, without traffic ATPase ATP hydrolysis, by substrate-induced, reversible bridging of the IM translocase to the OM export pore. Keywords: bacterial secretion mechanism/mitochondrial import/in vivo protein translocation/TolC membrane pore/traffic ATPase

Catalase-integrated hyaluronic acid as nanocarriers for enhanced photodynamic therapy in solid tumor. ACS Nano, 13(4), 4742-4751.

A cosmid library was prepared from a partial BamHJ digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons TnSOl and Tn2l. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADPribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.Bacillus sphaericus is an aerobic spore-forming Bacillus species, several strains of which are pathogenic for mosquito larvae (13). B. sphaericus SSII-1 was isolated in 1973 from infected mosquito larvae collected in India (36). This strain was considerably more toxic than the other B. sphaericus strains known at that time. Early studies indicated that the toxin was retained within or attached to the bacterial cells and that the toxic activity was markedly unstable. Storage of cells under refrigeration or heating at 80°C resulted in the loss of activity. Toxin production occurred predominantly in the vegetative phase of growth before the onset of sporulation (27,29).Further studies on B. sphaericus SSII-1 declined rapidly with the discovery of strains, such as 1593 (36a), 2362 (40), and 2297 (41), which had higher toxicities. In contrast to strain SSII-1, these strains develop relatively stable, high toxicity at the onset of sporulation (7). To date, two types of toxin have been characterized in the highly toxic strains: a pair of proteins of 51.4 and 41.9 kDa associated with the parasporal crystal (3,5,19) and a toxin of 110 kDa related to a 125-kDa surface layer protein (5, 6). Genes encoding the 51.4-and 41.9-kDa proteins were shown to be absent from strain SSII-i (4). In the same study, Baumann et al. showed that sequences inserted in their group C clones were also absent from strain SSII-1. The group C clones were later shown to contain sequences corresponding to the gene encoding the 125-kDa protein (6). Davidson (12) Construction of the cosmid library. To prepare total genomic DNA from B. sphaericus SSII-1, we lysed cells in the presence of 1% sodium dodecyl sulfate and 0.1 mg of proteinase K per ml. DNA was purified from the lysate by the method of Ausubel et al. (2). In brief, hexadecyltrimethyl ammonium bromide was added, and the mixture was incubated at 65°C for 20 min prior to extraction with chloroformisoamyl alcohol. DNA was precipitated with ethanol from the aqueous phase and purified by banding on a cesium chloride gradient coqtaining ethidium bromide. DNA was partially digest...

Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial–Mesenchymal Transition (EMT). EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM) probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor) is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor). Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.