Evaluation of a new model to detect bladder carcinogens or co-carcinogens; results obtained with saccharin, cyclamate and cyclophosphamide.

A sensitive rat model has been designed to detect potential weak bladder carcinogens or co-carcinogens. The test compound is given to animals which have received a single initiating, but non-carcinogenic, dose of N-methyl-N-nitrosourea (MNU). The model has been used to investigate two compounds currently under suspicion as weak bladder carcinogens, namely sodium saccharin and sodium cyclamate, and one compound known to be cytotoxic but not carcinogenic for the bladder epithelium namely cyclophosphamide. For comparison, these three compounds were also tested as solitary carcinogens in animals not pre-treated with MNU. At the very high dose levels used, sodium saccharin and sodium cyclamate were weak solitary carcinogens producing 4/253 and 3/228 bladder tumours respectively, and the first of these tumours did not appear for more than 80 weeks. When tested in the MNU/rat model more than half the animals receiving either sodium saccharin or sodium cyclamate developed bladder tumours from 10 weeks onwards. By contrast, cyclophosphamide failed to produce any tumours when tested either as a solitary carcinogen or in the MNU/rat model. It must be emphasized that the doses of saccharin and cyclamate used were far higher than those consumed by man, including diabetics, and these results should not be directly extrapolated to man without careful consideration of many other factors including negative epidemiological findings. The theoretical basis of the model is discussed and also the relevance, in terms of environmental human exposure, of detecting compounds which have a synergistic effect with other known bladder carcinogens. It appears that this model can be used to detect a carcinogenic or co-carcinogenic potential in compounds which are organotropic for the bladder more rapidly and with fewer animals than if the compounds are tested as solitary carcinogens by more conventional methods. It is suggested that it could be used to detect those compounds which require further investigation.     

Alkylation of cytosine and 5-hydroxymethyl-cytosine by methyl methanesulphonate and N-methyl-N-nitrosourea: its relevance to mutagenesis.

The reaction of cytosine and 5-hydroxymethyl-cytosine (OHMeCyt) with a variety of monofunctional alkylating agents has been investigated to evaluate further the possible role of cytosine alkylation in mutagenesis and the possibility that the immunity of T-even phages to mutation by methyl methanesulphonate (MMS) was due to the unreactivity of OHMeCyt towards this agent. Both cytosine and OHMeCyt reacted equally well with the methylating agents MMS and N-methyl-N-nitrosourea (MNU) affording 6% and less than 1% respectively of the 3-substituted derivative. No product was isolated following subjection of the bases to reaction with ethyl methane-sulphonate (EMS), N-ethyl-N-nitrosourea (ENU) or iso-propyl methane-sulphonate (iPMS).     

Physiological and biochemical changes associated with cotton fibre development. II. Auxin oxidising system

Changes in IAA oxidase, and in cytoplasmic and ionically wall-bound peroxidase activities were studied in the developing fibres of three cotton cultivars ( Gossypium hirsutum L. cv. Gujarat-67, cv. Khandwa-2 and G. herbaceum L. cv. Digvijay), designated as long, medium and short staple cultivars, respectively. In all the three cultivars IAA oxidase activity was low during the fibre elongation phase, while the activity increased significantly during the secondary thickening phase. The increase in IAA oxidase activity in the three cultivars showed close correspondence with their respective total period of elongation. No relationship between cytoplasmic peroxidase activity and fibre development was discernible. The ionically bound wall peroxidase activity, however, recorded low levels during the elongation phase and higher levels during the secondary thickening phase. The role of wall peroxidase in cessation of elongation growth is discussed.     

Acute fine structural changes in rat hepatocytes induced by a single large dose of 2-acetylaminofluorene.

Male rats were given a single intragastric dose of 2-acetylaminofluorene, 600 mg/kg body weight, killed at intervals up to 14 days after treatment, and their hepatic tissue examined by electron microscopy. The early cytoplasmic lesion produced in hepatocytes by lower doses, consisting of perinuclear glycogen pooling, peripheral displacement of organelles and pyknosis, was delayed for several days. Among the changes which appeared to be independent of this lesion were disorganization and decrease of the granular endoplasmic reticulum and abnormalities of the bile canaliculi. These changes were similar to those which are seen during chronic exposure to 2-AAF and other hepatocarcinogens and in hepatic cell tumours.     

Effects of a single dose of menadione on the intestinal calcium absorption and associated variables

The effect of a single large dose of menadione on intestinal calcium absorption and associated variables was investigated in chicks fed a normal diet. The data show that 2.5 micro mol of menadione/kg of b.w. causes inhibition of calcium transfer from lumen-to-blood within 30 min. This effect seems to be related to oxidative stress provoked by menadione as judged by glutathione depletion and an increment in the total carbonyl group content produced at the same time. Two enzymes presumably involved in calcium transcellular movement, such as alkaline phosphatase, located in the brush border membrane, and Ca(2+)- pump ATPase, which sits in the basolateral membrane, were also inhibited. The enzyme inhibition could be due to alterations caused by the appearance of free hydroxyl groups, which are triggered by glutathione depletion. Addition of glutathione monoester to the duodenal loop caused reversion of the menadione effect on both intestinal calcium absorption and alkaline phosphatase activity. In conclusion, menadione shifts the balance of oxidative and reductive processes in the enterocyte towards oxidation causing deleterious effects on intestinal Ca(2+) absorption and associated variables, which could be prevented by administration of oral glutathione monoester.     

Photoinduction kinetics of electrical potential in a single chloroplast as studied with micro-electrode technique

1. Using single chloroplasts of Peperomia metallica the kinetics of light-induced potential changes were studied. Three kinetic components (the initial fast rise, the decay in the light and the decay in the dark) were found to be characterized by time constants 4, 220 and 60 ms, respectively at light intensity 5000 lx and temperature 18 °C. After flash excitation the potential kept on rising for about 10 ms. Cooling of the medium down to 5 °C had no effect on the duration of potential rise after the flash.2. Variations in the medium temperature in the range 2–23 °C had little effect on photoresponse magnitude but resulted in significant acceleration of decay in the light.3. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (5 · 10^?6 M) resulted in suppression of the magnitude of the photoresponse but was not accompanied by any change in the rate of initial rise of potential. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-inhibited photoresponse could be restored and even enhanced by subsequent addition of N-methylphenazonium methosulfate (10^?4 M). N-Methylphenazonium methosulfate essentially influenced the time course and light-intensity curves of photoresponse.4. The chloroplast photoresponses were of different time-courses when elicited by red (640 nm) or far red (712 nm) light. This fact as well as an enhancement effect of combined illumination by two intermittent light beams indicate on the interaction of two photosynthetic pigment systems when the photoelectric response was formed.5. An imposed electrical field resulted in stimulation or suppression of chloroplast photoresponse depending on the polarity of the field. No indications for the existance of “reversal potential” for photoelectric response were obtained.6. A kinetic scheme of photoresponse formation is proposed, which includes two sequential photochemical reactions of photosynthesis.     

Metabolic responses to catecholamines in dogs injected with a single dose of triiodothyronine.

Lipolytic and glycogenolytic responses to catecholamine infusions were studied in resting dogs before and 20 h following administration of a single dose (0.1 mg/kg) of triiodothyronine (T3). In the dogs pretreated with T3 much higher increases in the plasma FFA concentration were found both during noradrenaline and adrenaline infusions in comparison with control experiments. Adrenaline-induced increases in blood LA and glucose levels were also significantly higher in T3-pretreated dogs than in controls. The blockade of beta-adrenergic receptors with propranolol prevented the increases in blood FFA and LA concentrations during subsequent adrenaline infusion. Phentolamine -- the alpha-adrenergic blocking agent -- infused to the T3-pretreated dog inhibited the adrenaline-induced rise in blood glucose level. The observed changes in the metabolic responses to catecholamines induced by triiodothyronine pretreatment indicate that at least in the dog this hormone potentiates both the lipolytic and glycogenolytic effects of catecholamines acting on appropriate adrenergic receptors.     

Genomic changes associated with antigenic variation of visna virus. II. Common nucleotide sequence changes detected in variants from independent isolations

Naturally occurring antigenic variants of visna virus isolated from two persistently infected sheep have been compared using RNAase T₁ fingerprint analysis of the viral RNAs. All the viruses examined have oligonucleotide changes in the 3′-terminal region of the viral genome and many of the alterations are common among the variants of both sheep. There is progressive accumulation of point mutations in the 3′-terminal region of antigenically altered viruses isolated from persistently infected animals. In addition, antigenic variants isolated independently from another sheep contain the same altered oligonucleotides; the nucleotide sequences of these oligonucleotides are identical. These data strongly suggest that the alterations observed in the nucleotide sequence of the variants are related to the change in their antigenic character.     

Primary reactions of Photosystem II at low pH. I. Prompt and delayed fluorescence

Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 μs, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 μs and was practically irreversible. The back reaction had a pH dependent half time: about 270 μs at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH.     

Environmental and ecological changes associated with a marina

Anthropogenic modifications to waterways are common and their ecological consequences must be understood to effectively conserve local biodiversity. The facilitation of recreational boating activities often requires substantial alteration of natural areas, however the environmental and ecological consequences of such alterations are rarely described in the scientific literature. In this study, ecological and physico-chemical conditions were investigated in a recreational boating marina, located inside a marine park on the south-east coast of Australia. Recruitment panels were deployed for 8?weeks both inside and outside the marina, and differences in the composition of the developing fouling communities were observed. The recruitment of taxa, which often have short-lived larvae, was increased inside the marina (bryozoans, spirorbids and sponges) while the recruitment of taxa, which often have longer-lived larvae, was reduced or absent (barnacles, solitary ascidians and non-spirorbid polychaetes). Differences were also observed in environmental conditions inside the marina cf. directly outside. The marina environment had higher turbidity, temperature and pH along with higher concentrations of lead and copper in suspended sediments, while flow rates and trapped sediment loads were reduced inside the marina. The differences observed in the study suggest that there may be marked environmental changes associated with marina developments. The potential ecological consequences of these changes should be a primary consideration during the planning process, particularly for developments in locations of notable ecological value.     

Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms

We previously demonstrated that chronic treatment with the mixed endothelin A and B (ET_A and ET_B) receptor blocker bosentan improved isolated working heart function in streptozotocin (STZ) diabetic rats. Endothelin-1 (ET-1) peptide levels, ET-1 mRNA and ET_A and ET_B receptor mRNA were all increased in diabetic hearts, but were unaffected by bosentan treatment, indicating that the beneficial effects of bosentan on heart appear to be on downstream effectors of ET-1 and ET receptors rather than the ET-1 system itself. Stimulation of ET-1 receptors leads to increased activation of protein kinase C (PKC), which is associated with PKC translocation from the cytosol to the membrane. Persistent activation of specific PKC isoforms has been proposed to contribute to diabetic cardiomyopathy. The purpose of this study was to determine whether chronic treatment with bosentan influences the activation of PKC isoforms in hearts from diabetic rats. Male Wistar rats were divided into four groups: control, bosentan-treated control, diabetic, and bosentan-treated diabetic. Diabetes was induced by the intravenous injection of 60 mg/kg streptozotocin. One week later, treatment with bosentan (100 mg/kg/day) by oral gavage was begun and continued for 10 weeks. The heart was then removed, homogenized, separated into soluble (cytosolic) and particulate (membrane) fractions and PKC isoform content in each fraction was determined by Western blotting. PKC α, β2, δ, ε and ζ were all detected in hearts from both control and diabetic rats. However, no change in the levels or distribution between the soluble and particulate fractions of any of these isoforms could be detected in chronic diabetic hearts compared to control, whether untreated or treated with bosentan. These observations indicate that bosentan does not improve cardiac performance in STZ diabetic rats by affecting the activation of PKC isoforms.     

Changes in gene expression associated with stable drug and radiation resistance in small cell lung cancer cells are similar to those caused by a single X-ray dose

Small cell lung cancer (SCLC) initially responds well to chemotherapy and fractionated radiotherapy, but resistance to these treatments eventually develops in the vast majority of cases. To understand how resistance develops in the H69 SCLC cell line, we compared the changes in gene expression associated with 37.5 Gy fractionated X-ray treatment that produced the stable radiation- and drug-resistant H69/R38 cell subline to the changes associated with a single 4- or 8-Gy X-ray treatment. Gene expression was determined by suppression subtractive hybridization combined with Northern blot analysis and two-dimensional (2D) protein electrophoresis. Stable radiation and drug resistance was associated with coordinate changes in the expression of genes of the cytoskeleton, protein synthesis, cell cycle, redox/stress and metabolic pathways. The pattern of these changes was remarkably similar to the changes seen 24 h after a single X-ray treatment of the H69 cells but differed from the changes in expression associated with a single X-ray treatment of the resistant H69/ R38 cells. Stable radiation and drug resistance may be caused by the constitutive expression of those genes transiently expressed by sensitive cells in response to a single X-ray dose. The repeated treatments received during fractionated irradiation may promote the change from a transient to a constitutive pattern of gene expression.     

The synergistic inhibition of breast cancer proliferation by combined treatment with 4EGI-1 and MK2206

Cap-dependent translation is a potential cancer-related target (oncotarget) due to its critical role in cancer initiation and progression. 4EGI-1, an inhibitor of eIF4E/eIF4G interaction, was discovered by screening chemical libraries of small molecules. 4EGI-1 inhibits cap-dependent translation initiation by impairing the assembly of the eIF4E/eIF4G complex, and therefore is a potential anti-cancer agent. Here, we report that 4EGI-1 also inhibits mTORC1 signaling independent of its inhibitory role on cap-dependent translation initiation. The inhibition of mTORC1 signaling by 4EGI-1 activates Akt due to both abrogation of the negative feedback loops from mTORC1 to PI3K and activation of mTORC2. We further validated that mTORC2 activity is required for 4EGI-1-mediated Akt activation. The activated Akt counteracted the anticancer effects of 4EGI-1. In support of this model, inhibition of Akt potentiates the antitumor activity of 4EGI-1 both in vitro and in a xenograft mouse model in vivo. Our results suggest that a combination of 4EGI-1and Akt inhibitor is a rational approach for the treatment of cancer.     

Trophic niche changes associated with habitat fragmentation in a Neotropical bat species

Habitat fragmentation could alter ecological traits including species trophic habits. Here, we used carbon and nitrogen stable isotope ratios to establish differences in isotopic niche width and food resource use between forest fragments and the continuous forest for the phyllostomid frugivorous bat Artibeus lituratus. Using mist nests, we captured bats from two forest fragments and two sites in continuous forest, and sampled from each individual captured three body tissues with contrasting turnover rates (skin, muscle, and liver). Samples were collected between February and March (austral summer) and between August and September (austral winter). In addition, in each sampling site and season we collected potential food resources (fruits and insects) consumed by our A. lituratus. Our findings indicate that A. lituratus had a predominantly omnivorous diet, with high consumption of insects during summer in forest fragments. The increasing consumption of insects in these fragments seems to have led to a wider isotopic niche, in relation to the continuous forest. Because A. lituratus is typically a seed disperser, changes in trophic habits in the forest fragments from frugivory to insectivory may diminish their role in forest regeneration. Abstract in Portuguese is available with online material.