The filamentous type III secretion translocon of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella, is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.

Structural studies on D2 dopamine receptors: mutation of a histidine residue specifically affects the binding of a subgroup of substituted benzamide drugs.

A histidine residue (His394) that is likely to be located in the ligand-binding region of the D2 dopamine receptor has been mutated to a leucine (Leu394), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+ sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His394 residue with these compounds.

The value of percutaneous transluminal angioplasty of the profunda femoris artery in threatened limb loss and intermittent claudication.

Percutaneous transluminal angioplasty (PTA) has been performed on 29 profunda femoris artery stenoses in 26 limbs of 25 patients. Seventy per cent of the 27 atheromatous stenoses involved the origin or proximal 4 cm of the profunda. One patient had two strictures of a common femoral-profunda femoris vein graft. All had total superficial femoral or femoropopliteal occlusion (median length 29.5 cm, range 4-47 cm). The 13 patients presenting with threatened limb loss were significantly older than the remainder, who had disabling intermittent claudication (P = 0.03), and had twice the incidence of diabetes mellitus. They also had significantly fewer calf vessels patent compared with the claudicants (P = 0.008). The approaches used for profunda PTA included ipsilateral antegrade common femoral (19), ipsilateral retrograde profunda (3), cross-over technique (2), antegrade profunda (1) and brachial cutdown (1). Profunda PTA was technically successful at 26 sites (89.7%), partially successful at one, and failed at two. Concomitant PTA was successful at eight of 10 sites in eight patients. Complications requiring surgery occurred in two (7.7%). The median follow up was 17.5 months (range 1-62 months). Of the 12 limb salvage patients who underwent a technically successful profunda PTA, six required no further intervention, three subsequently underwent bypass grafting and three had an inevitable amputation, the level of amputation having been lowered in one of the patients. Nine claudicants improved symptomatically after technically successful profunda PTA; three underwent an operative procedure. Eight (61.5%) of the limb salvage group have now died, compared with two (15.4%) of the claudicants. Profunda femoris PTA is an effective alternative to profundaplasty in patients with femoropopliteal occlusive disease and may obviate the need for bypass surgery or amputation.

Computed tomography (CT) and ultrasound (US) guided core biopsy in the management of non-Hodgkin's lymphoma.

Histological examination of adequate biopsy specimens is fundamental to the management of patients with non-Hodgkin's lymphoma (NHL). A practical alternative to open biopsy, provided enough tissue can be obtained, has obvious advantages, especially if the lesion in question is deep seated, and might call for laparotomy or thoracotomy. Core biopsy with computed tomography (CT) or ultrasound (US) guidance may be such an alternative, particularly when a spring-loaded firing device is used. Thirty-four biopsies were performed in 26 patients with known or suspected NHL. A primary histological diagnosis was made in 7/7 (six NHL, one seminoma). Relapse was confirmed in 15/15 patients overall. In patients with follicular NHL, 8/15 biopsies showed progression to high grade histology. Biopsies were also performed to assess the nature of residual abnormalities after treatment and to obtain fresh tissue for immunocytochemistry. Tissue was obtained in all cases and a further procedure (two laparotomies, one second needle biopsy) was required on only three occasions. The procedure was well tolerated and there were no complications. This technique is therefore a valuable alternative to more invasive surgical procedures and may be of major benefit in the management of NHL.

Liposarcoma: a ten year experience.

A retrospective study of all patients presenting with liposarcoma at Southend General Hospital between the years 1970 and 1979 is presented. There were 13 patients in the group treated with various combinations of surgery and radiotherapy. The histology has been reviewed: 7 patients had myxoid tumours, 2 well differentiated and 4 pleomorphic. The patients were followed up for between 18-109 months with 7 patients followed for more than 5 years. Local recurrence was seen only in myxoid tumours whereas pleomorphic tumours showed a high incidence of distant metastases. The length of survival was found to correlate with the histology, pleomorphic tumours being associated with the shortest survival. The series illustrates that the histological type of liposarcoma predicts the pattern of behaviour of the tumour and is the major determinant in prognosis.