Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

Partial purification and characterization of cysteine proteinases from various developmental stages of Paragonimus westermani

1. During development of Paragonimus westermani, larvae develop during migration within the host, and adult worms feed on pulmonary tissues, causing significant pathology in the mammalian host. In this report acidic extracts of various developmental stages (metacercariae and worms at one, two and three months of development) were examined for cysteine proteinase activity. 2. A soluble thiol-dependent proteinase activity with a native molecular weight of approximately 20,000 was isolated and partially purified. 3. The enzymes purified from the various developmental stages of the parasite had maximal activity at acidic pH and showed inhibitor susceptibilities similar to the vertebrate acidic cysteine proteinases. 4. Enzymatic activity was stable at pH 5.0 for at least two days when stored at 4 degrees C. 5. It is suggested that these enzymes may be involved in the nutrition of these parasites and/or during penetration and lysis of the tissues.     

Ecotoxicity of soils contaminated with industrial and domestic wastewater in western shenyang,China

Soil samples were collected from 7 sites in the up-, mid-and down-reach along and nearby the wastewater irrigation channel, western Shenyang of China. The concentrations of selected pollutants (mineral oil, PAHs - polycycle aromatic hydrocarbons and Cd) were determined by UV spectrometer, HPLC and AAS (atomic adsorption spectrometer) spectrometer, respectively. Toxicity effects of soils were evaluated by seedling emergence test with root length of wheat as the end-point and by earthworms test with the mortality rate and inhibition rates of body weight as endpoints. Results showed accumulation of pollutants for most soils with concentration of 200.2 mg.kg⁻¹∼1600 mg.kg⁻¹ for mineral oil, 0.33 mg.kg⁻¹∼1.81 mg.kg⁻¹ for Cd and 900.16 mg.kg⁻¹ ∼ 2737.91 mg.kg⁻¹ for PAHs. The inhibition rates of root elongation were from −20% up to 40 %, and mortality rates of earthworms ranged from 0%∼40% from the exposure period of two weeks to eight weeks by sampling interval of two weeks, the inhibition rates of earthworm growth were from −19.36% to 34.53%, showing effects of stimulation at 2 weeks to an increasing effects of inhibition at 4, 6 and 8 weeks, respectively. Mortality rates correlated with the loss of body weight of earthworms.

This study indicated the potential risk of pollutants of environmental low content in soil by the determination of selected chemicals combined with toxicity indexes.

     

Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion

A new beta-lactam-inducible penicillin-binding protein (PBP) that has extremely low affinity to penicillin and most other beta-lactam antibiotics has been widely found in highly beta-lactam(methicillin)-resistant Staphylococcus aureus (MRSA). The gene for this protein was sequenced and the nucleotide sequence in its promoter and close upstream area was found to show close similarity with that of staphylococcal penicillinase, while the amino acid sequence over a wide range of the molecule was found to be similar to those of two PBPs of Escherichia coli, the shape-determining protein (PBP 2) and septum-forming one (PBP 3). Probably the MRSA PBP (Mr 76462) evolved by recombination of two genes: an inducible type I penicillinase gene and a PBP gene of a bacterium, causing the formation of a beta-lactam-inducible MRSA PBP.     

Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

Final report of the Netherlands State Commission on Euthanasia: an English summary

An unofficial summary is provided of the Final Report of the Netherlands State Commission on Euthanasia, submitted to the Minister of Welfare, Health and Cultural Affairs and the Minister of Justice on 19 Aug 1985. It describes the establishment of the State Commission, its working methods, and its recommendations for changes in the Criminal Code with regard to euthanasia and assisted suicide. The Commission proposes that the Code be amended so that the intentional termination of another person's life at the latter's request would not be an offense if carried out by a doctor on a patient who is "in an untenable situation with no prospect of improvement." It makes recommendations regarding minors, the mentally handicapped, and prisoners, and regarding procedures for funeral arrangements and death certificates, the noninvolvement of parties other than patients and physicians in decision making, and the preparation and dispensing of drugs designed to terminate life.