High resolution structure of BipD: an invasion protein associated with the type III secretion system of Burkholderia pseudomallei.

Burkoldheria pseudomallei is a Gram-negative bacterium that possesses a protein secretion system similar to those found in Salmonella and Shigella. Recent work has indicated that the protein encoded by the BipD gene of B. pseudomallei is an important secreted virulence factor. BipD is similar in sequence to IpaD from Shigella and SipD from Salmonella and is therefore likely to be a translocator protein in the type-III secretion system of B. pseudomallei. The crystal structure of BipD has been solved at a resolution of 2.1 A revealing the detailed tertiary fold of the molecule. The overall structure is appreciably extended and consists of a bundle of antiparallel alpha-helical segments with two small beta-sheet regions. The longest helices of the molecule form a four-helix bundle and most of the remaining secondary structure elements (three helices and two three-stranded beta-sheets) are formed by the region linking the last two helices of the four-helix bundle. The structure suggests that the biologically active form of the molecule may be a dimer formed by contacts involving the C-terminal alpha-helix, which is the most strongly conserved part of the protein. Comparison of the structure of BipD with immunological and other data for IpaD indicates that the C-terminal alpha-helix is also involved in contacts with other proteins that form the translocon.

Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 angstroms resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 angstroms.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis.

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the P1 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

Analysis of FasL and TRAIL induced apoptosis pathways in glioma cells.

FasL and TNF-related apoptosis-inducing ligand (TRAIL) belong to a subgroup of the TNF superfamily which induce apoptosis by binding to their death domain containing receptors. In the present study we have utilized a panel of seven cell lines derived from human malignant gliomas to characterize molecular pathways through which FasL and TRAIL induce apoptosis in sensitive glioma cells and the mechanisms of resistance in cell lines which survive the death stimuli. Our findings indicate that FADD and Caspase-8 are essential for FasL and TRAIL mediated apoptosis in glioma cells. One sensitive cell line (D270) can be protected from FasL and TRAIL induced death by anti-apoptotic Bcl-2 family members while another (D645) cannot, implying that these lines may represent glioma examples of type II and type I cells respectively. For the first time we demonstrate resistance to FasL but not to TRAIL within the one glioma cell line. Furthermore, we report distinct mechanisms of resistance within different glioma lines, including downregulation of Caspase-8 in U373MG. Cycloheximide sensitized four of the resistant cell lines suggesting the presence of labile inhibitors. None of the known apoptosis inhibitors examined accounted for the observed resistance, suggesting novel inhibitors may exist in glioma cells.

A novel, rapid, computerized method for quantitation of neuronal damage in a rat model of stroke.

Determination of extent of infarction in animal models of cerebral ischemia is most commonly achieved by either classical histology (thionin staining) and light microscopy or staining with 2,3, 5-triphenyltetrazolium chloride (TTC). These techniques have limitations and we now describe a novel technique and its validation for assessment of the neuroprotective activity of AM-36, a novel arylalkypiperazine compound with combined antioxidant and sodium channel blocking activity. AM-36 (1.8 mg/kg i.p.) or vehicle, was administered 30 min, 24 and 48 h after endothelin-1-induced middle cerebral artery occlusion in conscious rats. Rats were killed at 72 h, brains removed and frozen in liquid nitrogen prior to coronal sectioning. Using a simple apparatus relying on basic principles of light propagation and a computerised image analysis system, ischemic damage in unstained slide-mounted sections was clearly visualised and measured. AM-36 significantly reduced the area of infarct in both cortex and striatum. The method was verified by thionin staining, and light microscopy. Linear regression analysis showed a highly significant correlation between methods at 72 h for infarct area in the cortex and striatum. Highly significant correlations between methods were found at 3 and 24 h after ischemia. Our method quickly and clearly delineates areas of damage in a manner superior to conventional staining methods.

Delayed treatment with AM-36, a novel neuroprotective agent, reduces neuronal damage after endothelin-1-induced middle cerebral artery occlusion in conscious rats.

BACKGROUND AND PURPOSE: AM-36 is a novel arylalkylpiperazine with combined antioxidant and Na(+) channel blocking actions. Individually, these properties have been shown to confer neuroprotection in a variety of in vitro and in vivo animal models of stroke. Preliminary studies have shown that AM-36 is neuroprotective in vivo. The purpose of the present study was to assess the neuroprotective and behavioral outcome after delayed administration of AM-36 in an endothelin-1-induced, middle cerebral artery model of cerebral ischemia in conscious rats. METHODS: Conscious male hooded Wistar rats were subjected to middle cerebral artery occlusion by perivascular microinjection of endothelin-1 via a previously implanted cannula. AM-36 (6 mg/kg IP) or vehicle was administered intraperitoneally 30, 60, or 180 minutes after middle cerebral artery occlusion. Functional outcome was determined 24, 48, and 72 hours after stroke by neurological deficit score, motor performance, and sensory hemineglect tests. Rats were killed at 72 hours, and infarct area and volume were determined by histology and computerized image analysis. RESULTS: Endothelin-1-induced middle cerebral artery occlusion resulted in marked functional deficits and neuronal damage. AM-36 significantly reduced cortical damage when administration was delayed until 30, 60, or 180 minutes after stroke. Interestingly, neuronal damage was time-dependently reduced, with the greatest protection found when AM-36 was administered 180 minutes after stroke. Striatal damage was significantly reduced after treatment with AM-36 at 180 minutes after stroke. Functional outcome paralleled histopathology. Rota-rod performance, sensory hemineglect, and neurological deficit scores returned to preischemia levels in AM-36-treated rats by 72 hours after stroke when administration was delayed by 180 minutes after stroke. CONCLUSIONS: AM-36 potently protects against both neuronal damage and functional deficits even when administered up to 180 minutes after induction of stroke. In fact, the greatest protection was found when administration was delayed by 180 minutes after stroke. The possible mechanisms of action of AM-36 are discussed. The present findings suggest that AM-36 may have great promise in the acute treatment of human stroke.

Umbilical port metastasis from gallbladder carcinoma after laparoscopic cholecystectomy.

A case of gallbladder carcinoma is presented where metastatic tumour developed at the abdominal wall port site following laparoscopic cholecystectomy.

Bile duct stents: is there an increased rate of complications in patients receiving chemotherapy?

The aim of this study was to determine whether palliative chemotherapy accelerates the rate of biliary stent occlusion, in patients with a malignant biliary obstruction. Such treatment can induce neutropenia and increase the risk of bacterial sepsis. Overgrowth of bacteria within the bile of patients receiving chemotherapy could accelerate the rate of stent occlusion. Retrospective analysis of treatment records for 80 consecutive patients with a diagnosis of adenocarcinoma arising from the pancreas, bile ducts or gall bladder was conducted. Two groups were identified, those with a biliary stent in situ (primary stent group: 47/80; 59%) at the time of referral and those without (no stent group: 33/80; 41%). The majority of patients went on to receive chemotherapy, 64% and 70% in the primary stent group and no stent group, respectively. The rate of febrile neutropenia was similar in the two groups (5% versus 7% of all chemotherapy cycles in the primary stent group and no stent group, respectively). The rate of stent occlusion was not significantly different between those exposed to chemotherapy (37%; 95% CI 20-54%) and those unexposed (39%; 95% CI 19-59%). Similarly, the mean duration of patency was not shortened by chemotherapy (105 days in the chemotherapy group versus 119 days in the non-chemotherapy group; P = 0.97, Mann-Whitney U-test). We conclude that there is no evidence of increased rate of bile duct-related complications in patients receiving chemotherapy. In particular, we find no indication for the use of prophylactic antibiotics.

A phase II study of continuous-infusion 5-fluorouracil with cisplatin and epirubicin in inoperable pancreatic cancer.

Carcinomas of the exocrine pancreas respond poorly to most chemotherapy regimens. Recently continuous infusional 5-fluorouracil (200 mg m-(2)day-1) with 3 weekly cisplatin (60 mg m-2) and epirubicin (50 mg m-2) (the ECF regimen) has proven to be an active regimen in gastric and breast cancer and consequently worthy of further study in pancreatic cancer. Thirty-five patients were treated with the ECF regimen as above, of whom 29 were evaluable for response and 32 were evaluable for toxicity. The mean age was 59 years (range 37-75). Sixteen patients had locally advanced disease at presentation and 19 had metastases. Objective tumour responses were documented in five (17.3%) patients who achieved a partial response; in 18 (62%) patients there were no change and six (20.7%) patients progressed on therapy. Patients with either stable disease or partial response had a significantly improved overall survival (median = 253 days) compared with patients who progressed (median = 170 days; P = 0.01). Grade 3/4 (WHO) toxicity (all cycles) included alopecia in 18 (56%) patients, nausea/vomiting in eight (25%) stomatitis in three (9%) and diarrhoea in seven (22%) patients, with rhinorrhoea and excessive lacrimation in one patient each. Neutropenic sepsis occurred in 13 cycles in ten patients, and there was one toxic death due to sepsis. There were eight other episodes of non-neutropenic sepsis requiring hospital admission. Fourteen patients (40%) experienced complications with their Hickman lines, including thrombotic episodes (six patients) or their line falling out (five patients). ECF can prolong survival in patients with locally advanced or metastatic pancreatic cancer who demonstrate a response or stabilisation of their disease. However, this is associated with considerable toxicity.

Toxicity of melaleuca oil and related essential oils applied topically on dogs and cats.

Cases of melaleuca oil toxicosis have been reported by veterinarians to the National Animal Poison Control Center when the oil was applied dermally to dogs and cats. In most cases, the oil was used to treat dermatologic conditions at inappropriate high doses. The typical signs observed were depression, weakness, incoordination and muscle tremors. The active ingredients of commercial melaleuca oil are predominantly cyclic terpenes. Treatment of clinical signs and supportive care has been sufficient to achieve recovery without sequelae within 2-3 d.

Pancreaticogastrostomy: the preferred operation for pain relief in chronic pancreatitis.

We present the results of surgery in 53 patients with intractable pain due to chronic pancreatitis associated with pancreatic duct dilatation. Using a limited mucosal to mucosal anastomosis over a silastic T tube the main pancreatic duct was drained in 33 patients into a Roux-en-Y jejunal loop (pancreaticojejunostomy, PJ) and in 20 patients into the stomach (pancreaticogastrostomy, PG). There was one postoperative death in the PJ group and none in the PG group. All patients were followed up for a minimum of four years. There was significantly greater pain relief in the PG group both at 1 (P less than 0.01) and 4 years (P less than 0.05) after surgery. We argue that PG is the operation of choice to relieve intractable pain in most patients with chronic pancreatitis associated with duct dilatation.