Bax targeting to mitochondria occurs via both tail anchor-dependent and -independent mechanisms.

Bax is a member of the Bcl-2 family that, together with Bak, is required for permeabilisation of the outer mitochondrial membrane (OMM). Bax differs from Bak in that it is predominantly cytosolic in healthy cells and only associates with the OMM after an apoptotic signal. How Bax is targeted to the OMM is still a matter of debate, with both a C-terminal tail anchor and an N-terminal pre-sequence being implicated. We now show definitively that Bax does not contain an N-terminal import sequence, but does have a C-terminal anchor. The isolated N terminus of Bax cannot target a heterologous protein to the OMM, whereas the C terminus can. Furthermore, if the C terminus is blocked, Bax fails to target to mitochondria upon receipt of an apoptotic stimulus. Zebra fish Bax, which shows a high degree of amino-acid homology with mammalian Bax within the C terminus, but not in the N terminus, can rescue the defective cell-death phenotype of Bax/Bak-deficient cells. Interestingly, we find that Bax mutants, which themselves cannot target mitochondria or induce apoptosis, are recruited to clusters of activated wild-type Bax on the OMM of apoptotic cells. This appears to be an amplification of Bax activation during cell death that is independent of the normal tail anchor-mediated targeting.

An intron 1 enhancer element mediates oestrogen-induced suppression of ERBB2 expression.

Overexpression of the ERBB2 gene in human breast cancer is associated with a poor prognosis and resistance to hormonal treatment and chemotherapy. Oestrogen receptor (ER) positive tumour-derived cell lines are known to express relatively low levels of ERBB2 protein under oestrogenic conditions, but markedly higher levels following withdrawal of oestrogens or administration of tamoxifen. Expression of the closely related ERBB3 gene, which co-operates with ERBB2 in cellular transformation, is now shown to respond to oestrogenic manipulation in a similar way, both responses being mediated largely by transcriptional changes. Six previously undescribed DNase I hypersensitive sites occur within the first intron of ERBB2 in cells that overexpress the gene. A 409 base pair DNA fragment containing one of these sites conferred ER dependent oestrogen inhibition on the ERBB2 promoter in two types of transient transfection assay. DNase I footprinting revealed four separate transcription factor binding sites within this fragment consistent with a role as a transcriptional enhancer. These findings implicate intron 1 sequences in the control of ERBB2 expression for the first time and demonstrate that one site within this region is involved in mediating the transcriptional response to oestrogens. Additionally, there is likely to be synergism between ERBB2 and ERBB3 signalling when both are overexpressed in response to oestrogen inhibition, thereby driving transformed cell behaviour.

Cofactor competition between the ligand-bound oestrogen receptor and an intron 1 enhancer leads to oestrogen repression of ERBB2 expression in breast cancer.

Overexpression of the ERBB2 proto-oncogene in breast tumours, which occurs in 25-30% of patients, correlates with poor prognosis. In oestrogen receptor (ER) positive breast epithelial cells oestrogens reduce ERBB2 mRNA and protein levels, an effect that is reversed in the presence of anti-oestrogens such as tamoxifen and ICI 182780. Our previous studies have shown that the major effect of oestrogen on ERBB2 expression is at the level of transcription and that this is mediated through a region within the ERBB2 first intron which can act as an oestrogen-suppressible enhancer in ER positive breast cells. In vitro footprinting of the smallest DNA fragment that retained full activity revealed four transcription factor binding sites. We report here that two of these sites are recognized by AP-2 proteins and the other two are bound by a variety of bZIP factors, including CREB and ATFI, with a major complex containing ATFa/ JunD. However, by using ER mutants it is clear that repression occurs essentially off the DNA. Indeed, the essential domain of the ER responsible for repression of the ERBB2 enhancer is a region termed AF2 which is required for the ligand-dependent association of non-DNA binding cofactors. We further demonstrate that one of these ER cofactors, SRC-1, can relieve oestrogen repression of the ERBB2 enhancer and conclude that these data fit with a model whereby the ER and the ERBB2 enhancer compete for this limiting, non-DNA binding cofactor. Thus, in oestrogenic conditions SRC-1 preferentially binds to the ER which effectively sequesters it thereby reducing enhancer activity, but in antioestrogenic media the cofactor is released from the ER and is therefore available to activate the ERBB2 enhancer.

Efficacy and toxicity of vinblastine, bleomycin, and methotrexate with involved-field radiotherapy in clinical stage IA and IIA Hodgkin's disease: a British National Lymphoma Investigation pilot study.

PURPOSE: To assess the efficacy and toxicity of vinblastine, bleomycin, and methotrexate (VBM) chemotherapy with involved-field radiotherapy in clinical stage IA and IIA Hodgkin's disease. PATIENTS AND METHODS: Thirty eligible patients with clinical stage IA or IIA Hodgkin's disease, at intermediate risk of relapse, were enrolled into a prospective multicenter pilot study. They received two cycles of VBM chemotherapy, followed by involved-field radiotherapy and then four further cycles of VBM. The median follow-up duration from the start of treatment is 30 months. RESULTS: All 26 patients with assessable disease showed an objective response after two cycles of VBM (nine complete responses, 17 partial responses). By the completion of treatment, 27 patients were in complete remission; two had stable residual masses, which have not progressed at 26 and 34 months of follow-up; and one patient who died of treatment-related sepsis was in complete remission at that time. Two relapses have occurred, 19 and 28 months after starting VBM. Cough and dyspnea developed in 14 of 30 patients, and were associated with impairment of pulmonary function tests. Three episodes of neutropenic sepsis were recorded. CONCLUSION: VBM with involved-field radiotherapy is an effective treatment for early Hodgkin's disease. However, the associated toxicity, both pulmonary and hematologic, is severe, making the regimen unsuitable for routine use.

Benzalkonium chloride exposure in cats: a retrospective analysis of 245 cases reported to the Veterinary Poisons Information Service (VPIS).

Benzalkonium chloride is commonly found in household products. This retrospective study examined 245 cases of feline exposure to benzalkonium chloride-containing products reported to the Veterinary Poisons Information Service (VPIS). A single route of exposure was reported in 188 cats (ingestion 126, skin 58, buccal 4); 57 cats had multiple routes. The common products involved were household antibacterial cleaners (43.6 per cent), household disinfectants (22.3 per cent) and patio cleaners (17.5 per cent). The most common signs were hypersalivation/drooling (53.9 per cent), tongue ulceration (40.4 per cent), hyperthermia (40.4 per cent) and oral ulceration (22.9 per cent). The mean time recorded for onset of the first clinical sign was 6.4 hours (range five minutes to 48 hours, median 4.5 hours, n=60), however, the VPIS was not contacted until 14.0 ± 13.2 hours after exposure (n=120). This figure also reflects the time of presentation. The most common treatments given were antibiotics (82.0 per cent), fluids (50.2 per cent), analgesia (45.3 per cent), gastroprotectants (31.0 per cent), dermal decontamination (24.1 per cent) and steroids (22.7 per cent). 13 cats (5.3 per cent) received syringe or nasogastric feeding. Of 245 cats, 12 (4.9 per cent) remained asymptomatic, 230 (93.9 per cent) recovered and three died (1.2 per cent). The time to recovery ranged from 1 to 360 hours (n=67) with a mean of 100.4 ± 82.0 hours (4.2 ± 3.4 days, median 72 hours).

Common questions in veterinary toxicology.

Toxicology is a vast subject. Animals are exposed to numerous drugs, household products, plants, chemicals, pesticides and venomous animals. In addition to the individual toxicity of the various potential poisons, there is also the question of individual response and, more importantly, of species differences in toxicity. This review serves to address some of the common questions asked when dealing with animals with possible poisoning, providing evidence where available. The role of emetics, activated charcoal and lipid infusion in the management of poisoning in animals, the toxic dose of chocolate, grapes and dried fruit in dogs, the use of antidotes in paracetamol poisoning, timing of antidotal therapy in ethylene glycol toxicosis and whether lilies are toxic to dogs are discussed.

Acute poisoning: initial management and sources of information.

Poisoning is responsible for 2-3 per cent of attendances to urban A&F. departments in the UK each year ( MacNamara et al 1996 ). It accounts for about 7 per cent of accidents in children under five years of age ( Consumer Safety Unit 1995 ). The National Poison Information Service (NPIS) deals with hundreds of emergency telephone enquiries every day. and the number of such calls has risen sharply in recent years. Figure 1 demonstrates the annual call load of NPIS (London). A&E nurses account for 42 per cent of these telephone enquiries.

Lipid infusion in the management of poisoning: a report of 6 canine cases.

Intravenous administration of lipid is a relatively new treatment in the management of toxicity from lipophilic compounds. It is used in human medicine in the treatment of toxicity from lipophilic local anaesthetics and cardiotoxic drugs and can result in dramatic improvement in clinical status. We present six cases of poisoning in dogs successfully treated with lipid infusion after ingestion of ivermectin (3), moxidectin (2) and baclofen (1). The dogs ranged in age from eight weeks to 14 years, and weighed 4-30 kg. Intravenous lipid therapy was started between six and eight hours and 22 hours after ingestion, and all the dogs responded well. In four dogs, there was clinical improvement within one hour; one had improved within two hours and the other within 4.5 hours of lipid administration. The only adverse effect of lipid infusion reported was mild swelling and pain after extravasation in one case which resolved with conservative management. All the dogs were discharged within 24-52 hours after exposure (7-46 hours after the start of lipid administration), and none developed any apparent sequelae.

Suspected metaldehyde slug bait poisoning in dogs: a retrospective analysis of cases reported to the Veterinary Poisons Information Service.

A retrospective analysis of telephone enquiries to the Veterinary Poisons Information Service found 772 cases with follow-up concerning suspected metaldehyde slug bait ingestion in dogs between 1985 and 2010. Half the enquiries occurred in the summer months. The amount and strength of the slug bait ingested was rarely known. In 56, cases the quantity consumed was estimated and was on average 229.6 grams of bait. Clinical signs developed in 77.3 per cent of dogs; common signs were convulsions, hypersalivation, twitching, hyperaesthesia, tremor, vomiting, hyperthermia and ataxia. Only 4.6 per cent of dogs developed hepatic changes, and only one developed renal impairment. The average time to onset of signs was 2.9 hours post-ingestion, with 50.3 per cent of dogs developing effects within one hour. Increased muscle activity (twitching, convulsions) lasted on average 15.2 hours. Recovery time was reported in 61 cases and occurred on average at 39.3 hours. Common treatments were gut decontamination, anticonvulsants, anaesthetics and intravenous fluids. Of the dogs that were treated with sedatives, 45.8 per cent required more than one sedative or anaesthetic agent. Methocarbamol was rarely used, probably due to unavailability. The outcome was reported in 762 dogs; 21.7 per cent remained asymptomatic, 61.7 per cent recovered and 16 per cent of dogs died or were euthanased. Where known (only six cases), the fatal dose of bait ranged from 4.2 to 26.7 g/kg (average 11.8 g/kg).

Canine adder bites in the UK: a retrospective study of cases reported to the Veterinary Poisons Information Service.

This retrospective study examined cases with follow-up reported to the Veterinary Poisons Information Service (VPIS) between September 1985 and December 2010. Most bites (69.2 per cent) occurred between April and July, particularly between 15:00 and 16:00 hours. Adder bites were more frequently reported in the south-east of England, particularly in Surrey. Swelling to the face and limbs was common, as was lethargy, depression, hyperthermia and tachycardia. About two-thirds of dogs developed both systemic and local effects, while a third developed local effects alone. Initial clinical effects usually occurred within two hours, with full recovery typically occurring five days after the bite. Antivenom was used in 55.9 per cent of cases and appeared to significantly reduce duration of oedema from an average of 94 to 47 hours. Adder bites can cause significant morbidity (97 per cent of dogs were symptomatic), but mortality is low (4.6 per cent died).