Integration of mammalian, microbial and drosophila procedures for evaluating chemical mutagens.

cis-Platinum(II)diamminodichloride (PDD), an anti-tumor agent, induced auxotrophic mutations in Escherichia coli, some of which were reverted to prototrophy by exposure to PDD, 2-aminopurine (2-AP), and N-methyl-N′-nitro-N-nitroguanidine (NTG), but not ICR derivatives. Similarly, various 2-AP-, NTG-, and ultraviolet light-induced auxotrophs were reverted to prototrophy by PDD. Some PDD-induced auxotrophs carried nonsense mutations and others could be phenotypically suppressed by growth with streptomycin. Although these findings suggest that PDD promotes base substitutions, this mutagen may also cause base subtractions because (like NTG)it induced, at reduced frequency, reversion to prototrophy of certain ICR-induced auxotrophs. Isomeric trans-platinum(II)diamminodichloride, which lacks anti-tumor activity, was an ineffective mutagen. Near-optimal conditions for PDD-induced mutagenesis entailed prolonged cultivation with low levels of mutagen where the frequency of forward mutation to auxotrophy was 10⁻³ and that of a selected trp isolate to prototrophy was 10⁻².     

Radioimmunoassay for plasma betamethasone 17-benzoate.

A sensitive radioimmunoassay for plasma betamethasone 17-benzoate has been developed. The antiserum used was obtained by immunizing rabbits with betamethasone 17-benzoate-21-hemisuccinate-bovine-serum-albumin conjugate. All of the endogenous steroids tested cross reacted less than 0.10%. A standard curve was established with a useful range from 0.05-5 ng. Reliability criteria were satisfactory. Measurement of plasma concentrations of betamethasone 17-benzoate was performed in patients and in rabbits following occlusive dressing of betamethasone 17-benzoate cream and gel base.     

The use of isochronal reference standards in phase and modulation fluorescence lifetime measurements

Errors in phase and modulation lifetime measurements observed with the only commercially available instrument are readily apparent when the Debye-Sears modulation tank is not perfectly tuned. Unfortunately, we have found that exact tuning was extremely difficult to achieve and maintain. We report that these errors could be reduced by using single-lifetime (homogeneous) reference standards whose fluorescence lifetime approximated that of the unknown sample (isochronal standards). A number of useful standards are suggested. In the proposed method, the phase shift and relative modulation of the sample emission are measured using the isochronal standard as a reference to determine the effective characteristics of the sinusoidal excitation. The importance of the improvement in accuracy accomplished by the proposed methods is illustrated by the accurate resolution of fluorescence lifetime heterogeneity for two known heterogeneous samples.     

Arabica coffee and olive oils mitigate malathion-induced nephrotoxicity in rat: In silico,immunohistochemical and biochemical evaluation

Malathion (MAL) is an organophosphate insecticide that disrupts the body's antioxidant system; it is one of the earliest organophosphate insecticides extensively used as dust, emulsion, and vapor control a wide variety of insect pests under different conditions. This experimentation aims to evaluate the influence of Arabica coffee oil and olive oil on MAL-induced nephrotoxicity in male rat. 6 sets bearing the same number of animals were applied to this experiment. Each set comprised 10 rats. The first set of rats was used as the control group; rats in the second set were exposed to MAL measured at 100 mg/kg body weight for 7 weeks. Animals in the third and fourth set were treated with 400 mg/kg body weight of Arabica coffee oil and olive oil, and 100 mg/kg body weight of MAL. The fifth, together with the sixth set, were fed with a similar proportion of Arabica coffee oil and olive oil as administered to the third set of rats. After the experimental duration, rats of group 2 showed severe biochemical alterations, including significant increases of creatinine, uric acids, and urea nitrogen (BUN), resulting in marked decreases in serum albumin values and total protein (TP). Severe histopathological and immunohistochemical alterations of kidney tissues were observed in exposed MAL-intoxicated rats. Administration of these oils reduced the detected biochemical, histopathological modifications caused by MAL intoxication. Two active ingredients in Arabica coffee oil (oleic acid) and olive oil (hydroxytyrosol) showed good cyclooxygenase-2 (COX 2) interaction. Moreover, oleic acid from coffee oil and olive oil exhibited impressive association with xanthine oxidase (XO). The current finding showed that coffee oil and olive oil could be appraised as possible and a likely deterrence component against nephrotoxicity brought about by MAL.     

The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.     

The induction of cell-mediated immunity and tolerance to insulin with insulin coupled to syngeneic lymphoid cells

The induction of delayed type hypersensitivity (DTH) and tolerance to DTH against bovine insulin in mice were explored. DTH was induced with insulin in complete Freund's adjuvant (CFA) and was assessed by ear swelling in vivo and by antigen-driven cell proliferation in vitro. Using the concept that thymus cell unresponsiveness is most easily accomplished via antigen on syngeneic membranes, tolerance was induced by iv injection of syngeneic lymphoid cells which had been coupled to insulin with carbodiimide. Mice tolerized with insulin-coupled cells and then sensitized with insulin-CFA had diminished ear swelling in vivo and decreased insulin-driven cell proliferation in vitro. This unresponsiveness was antigen specific but was also inconstant in degree with regard to suppression of ear swelling, most likely because of variability in coupling of insulin to cells. Proliferative responses were more uniformly suppressed, suggesting the possibility that two target cells were being tolerized. Thus, as with other proteins, the biologically active insulin can be used to induce tolerance.     

Structure of the Human Cation-Independent Mannose 6-Phosphate/IGF2 Receptor Domains 7–11 Uncovers the Mannose 6-Phosphate Binding Site of Domain 9

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