The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Osmotic regulation of transcription: induction of the proU betaine transport gene is dependent on accumulation of intracellular potassium

The proU locus, which encodes a high-affinity betaine transport system, and the kdp operon, which encodes a potassium transport system, are the principal osmoresponsive genes in Escherichia coli and Salmonella typhimurium. The kdp operon is known to be induced in response to changes in cell turgor. We have investigated the control of proU expression and shown that it differs from that of kdp in a number of fundamental ways. Rather than responding to changes in turgor, proU expression is principally determined by the intracellular accumulation of potassium ions. Potassium and betaine were shown to play distinct osmoprotective roles. Potassium serves as the principal osmoprotectant and is accumulated in response to low-level osmotic stress to restore turgor. As external osmolarity is increased to a level at which the corresponding increase in internal potassium concentrations is potentially deleterious to enzyme function, betaine (when available) is accumulated in preference to potassium. The different mechanisms of proU and kdp regulation reflect the different physiological roles of these two osmoprotectants.     

Interactions between Pisolithus tinctorius and its hosts: a review of current knowledge

 Pisolithus tinctorius (Pers.) Coker and Couch [Syn. = P. arhizus (Scop.: Pers.) Rauschert] (Pt) is a widespread ectomycorrhizal basidiomycete forming mycorrhizas with a variety of hosts. Developmental and functional aspects of the symbiosis are well documented and thus Pt has been adopted as a model organism for investigations of the molecular basis of ectomycorrhizal interactions. In this review of the current state of knowledge of interactions between Pt and its hosts we demonstrate that Pt displays much intraspecific heterogeneity of host specificity, physiology and the benefits the fungus can impart upon the host plant. It is not clear at present how far such heterogeneity reflects systematic segregation within Pt. Accepted: 20 May 1997     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Population‐based analysis of health care contacts among suicide decedents: identifying opportunities for more targeted suicide prevention strategies

The objective of this study was to detail the nature and correlates of mental health and non‐mental health care contacts prior to suicide death. We conducted a systematic extraction of data from records at the Office of the Chief Coroner of Ontario of each person who died by suicide in the city of Toronto from 1998 to 2011. Data on 2,835 suicide deaths were linked with provincial health administrative data to identify health care contacts during the 12 months prior to suicide. Sub‐populations of suicide decedents based on the presence and type of mental health care contact were described and compared across socio‐demographic, clinical and suicide‐specific variables. Time periods from last mental health contact to date of death were calculated and a Cox proportional hazards model examined covariates. Among suicide decedents, 91.7% had some type of past‐year health care contact prior to death, 66.4% had a mental health care contact, and 25.3% had only non‐mental health contacts. The most common type of mental health contact was an outpatient primary care visit (54.0%), followed by an outpatient psychiatric visit (39.8%), an emergency department visit (31.1%), and a psychiatric hospitalization (21.0%). The median time from last mental health contact to death was 18 days (interquartile range 5‐63). Mental health contact was significantly associated with female gender, age 25‐64, absence of a psychosocial stressor, diagnosis of schizophrenia or bipolar disorder, past suicide attempt, self‐poisoning method and absence of a suicide note. Significant differences between sub‐populations of suicide decedents based on the presence and nature of their health care contacts suggest the need for targeting of community and clinical‐based suicide prevention strategies. The predominance of ambulatory mental health care contacts, often close to the time of death, reinforce the importance of concentrating efforts on embedding risk assessment and care pathways into all routine primary and specialty clinical care, and not only acute care settings.     

Molecular identification of co-occurring Cortinarius and Dermocybe species from southeastern Australian sclerophyll forests

 The ability of restriction fragment length polymorphism (RFLP) analysis of the rDNA internal transcribed spacer (ITS) region to discriminate 10 co-occurring Cortinarius and Dermocybe species at a southeastern Australian sclerophyll forest site was assessed. Using the basidiomycete-specific primers ITS1F and ITS4B, some taxa were separated on the basis of individual RFLP patterns derived using the restriction endonucleases Hae III or Hinf I. Combined data from both endonucleases were, however, required to separate all taxa [Dermocybe austro-veneta Clel. (Moser & Horak), C. rotundisporus Clel. & Cheel, C. archeri Berk., C. sinapicolor Clel., C. violaceus (L.: Fr.) S.F.Gray, C. radicatus Clel. and four morphologically-distinct, but unidentified Cortinarius spp.]. ITS sequence comparisons confirmed that D. austro-veneta belongs in Dermocybe, that C. rotundisporus is correctly placed in subgenus Phlegmacium, and suggest that Australian C. violaceus collections are not conspecific with northern hemisphere C. violaceus. Accepted: 4 March 1999     

Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest

Fungi were isolated from the roots of 17 plant species from the families Apiaceae, Cunoniaceae, Cyperaceae, Droseraceae, Fabaceae-Mimosoideae, Lomandraceae, Myrtaceae, Pittosporaceae, Proteaceae and Stylidiaceae at a sclerophyll forest site in New South Wales, Australia. Internal transcribed spacer (ITS) restriction fragment length polymorphism (RFLP) and sequence comparisons indicated that the isolated fungi had affinities to a range of ascomycetes, basidiomycetes and zygomycetes. Four RFLP types had closest affinities to previously identified Helotiales ericoid mycorrhizal (ERM) or Oidiodendron spp. Isolates representing six RFLP types, which were variously isolated from all 17 plant species, formed ERM coils in hair root epidermal cells of Woollsia pungens (Ericaceae) under gnotobiotic conditions. Three of these isolates formed intercellular hyphae, intracellular hyphae and/or microsclerotia, which are typical of dark septate endophyte infection, in roots of Stylidium productum (Stylidiaceae), indicating an ability to form different types of association with roots of different hosts. Overall the data indicate that a broad range of plant taxa may act as repositories for ERM fungi in sclerophyll forest soil.