Acquired resistance to temsirolimus in human renal cell carcinoma cells is mediated by the constitutive activation of signal transduction pathways through mTORC2.

BACKGROUND: The objective of this study was to characterise the mechanism underlying acquired resistance to temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), in renal cell carcinoma (RCC). METHODS: A parental human RCC cell line, ACHN (ACHN/P), was continuously exposed to increasing doses of up to 20 µM of temsirolimus, and a cell line resistant to temsirolimus (ACHN/R), showing a sixfold higher IC50 than that of ACHN/P, was developed. RESULTS: Following treatment with temsirolimus, phosphorylation of S6 kinase in ACHN/P was markedly inhibited, whereas there was no detectable expression of phosphorylated S6 in ACHN/R before and after temsirolimus treatment. However, AKT and p44/42 mitogen-activated protein kinase (MAPK) were constitutively phosphorylated even after temsirolimus treatment in ACHN/R, but not in ACHN/P. There was no significant difference between the sensitivities of ACHN/P and ACHN/R to KU0063794, a dual inhibitor of mTOR complex 1 (mTORC1) and mTORC2. Similar sensitivities to temsirolimus in ACHN/P and ACHN/R could be achieved by additional treatment with specific inhibitors of AKT- and MAPK-signaling pathways. CONCLUSION: The activation of signal transduction pathways via mTORC2, but not via mTORC1, may have an important role in the acquisition of a resistant phenotype to temsirolimus in RCC.

The complete nucleotide sequence of a 23S rRNA gene from a blue-green alga, Anacystis nidulans.

The complete nucleotide sequence of a 23S rRNA gene from a blue-green alga, Anacystis nidulans, has been determined. This nucleotide sequence has 78% and 68% homologies with those of the tobacco chloroplast and Escherichia coli 23S rRNA genes, respectively. The 3'-terminal region of the A. nidulans 23S rRNA gene has strong homology with the chloroplast 4.5S rRNA.

Stimulation of SV40 DNA replication by the human c-myc enhancer.

In earlier studies we had shown that a transcriptional enhancer sequence exists about 2 kb upstream of the human c-myc gene. The core sequence necessary for enhancer activity was defined therein as a 21 bp nucleotide element, which also showed autonomous replicating activity [EMBO J. (1988) 7, 3135-3142; EMBO J. (1989) 8, 4273-4279]. Recently, several reports have substantiated the notion that transcription and replication can be concertedly regulated in a larger number of cases than expected. In this report, we took the simian virus 40 (SV 40) ori/promoter as a model system. The SV40 enhancer is known to enhance transcription from its ori/promoter, but to reduce its replication (probably due to a negative feedback). The SV40 enhancer was replaced by the c-myc enhancer core in order to see its effect upon SV40 DNA replication and transcription. The results showed that besides stimulating transcription, the c-myc enhancer promoted SV40 DNA replication in monkey CosI cells. Stimulation was only observed when the c-myc enhancer was inserted in the 'up-to-down' orientation to the SV40 promoter. The promoting function of the c-myc enhancer on DNA replication correlated with the transcriptional activation function, as determined by systematic point mutations introduced within the 21 bp core sequence.

Substrate specificity of bovine liver cytosolic neutral alpha-mannosidase activated by Co2+.

A cytosolic neutral alpha-mannosidase was purified from bovine liver. Its molecular weight was found to be 500,000 on gel filtration. The activity of the enzyme toward Man alpha 1-6-(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc-PA was increased 26-fold by preincubation with 1 mM Co2+. Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc was hydrolyzed by the enzyme to Man alpha 1-3Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc, which was further hydrolyzed to Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc. The rate of hydrolysis was 15-fold greater than that of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. This substrate specificity suggested that the enzyme could be involved in the degradation of oligomannose-type sugar chains with one GlcNAc residue released from glycoproteins by endo-beta-N-acetylglucosaminidase, and supported a pathway for glycoprotein catabolism via oligomannosyl glycans with one GlcNAc residue proposed on the basis of an earlier study on a cytosolic neutral alpha-mannosidase from Japanese quail oviduct [Oku, H. and Hase, S. (1991) J. Biochem. 110, 982-989].

A research to develop a predicting system of mammalian subacute toxicity. I. Prediction of subacute toxicity using the biological parameters of acute toxicities.

Predicting equations of subacute toxicity were developed by analyzing rat acute and subacute toxicity data of 56 chemicals of various structures. Minimum or 10% effect level in acute or subacute toxicity was estimated as a "biological parameter". Good regression equations were established between the geometrical means ("combined parameters") of any two of the parameters of acute and subacute toxicities and introduction of log P to the equations improved the correlations with a statistically significant multiple regression coefficient. The lowest predicted effect level of the subacute toxicity, which is selected from the data calculated by the above several correlations, can predict the upper limit of the no observed effect level. In recent years, for research and development of new chemical substances, it becomes one of the important factors that these have a lower environmental load in nature. Eventually, it becomes essential to evaluate not only their acute effects on human or environments, but also their chronic influences when they are to be exposed for a long period of time, and the cost for such verification is becoming a breaking factor for research and development. Thus, the development of a new technique which estimates the environmental load of a chemical substance including toxic effects on human with lower cost is now being attempted. For example, the development of in vitro new alternative methods using cultured cells, the utilization of a data base or software which relates mammalian and environmental toxicities and so on are internationally carried forwards. As for the latter, quantitative structure-activity relationship (QSAR) techniques have been applied practically to decide appropriate toxicity tests needed for regulatory purposes by EPA/TSCA, ITC/TSCA and FDA/FDAA in the USA. In addition, it was concluded recently in a joint meeting of EU and US-EPA that the approach by QSAR techniques was useful to specify the new chemical substances which are to be required toxicological examinations. In these QSAR techniques, however, while there are some considerations about common mechanisms of toxicity among chemicals with a similar structure (Congeneric chemicals, Congeners) for establishing of correlation formulas, for chemicals with various structures (Non-congeneric chemicals, Non-congeners) there often lack such common considerations. In addition, biological or physiological factors which are basic toxic indices are often ignored. In a previous study, we researched acute and subacute oral toxicities of industrial common chemicals in rats and reported the followings; 1) their subacute toxicological spectrum in target organs/tissues and morphologic changes was very limited and specific, 2) the important targets were liver, kidneys, blood (spleen) and stomach and these are considered the sites of dominant exposure due to the kinetics of chemical substances, 3) the morphological changes were hepatocellular hypertrophy, deposition of substance in renal tubules, extramedullary hematopoiesis in spleen and mucous lesion in stomach, and these are implying adaptation such as induction of drug-metabolizing enzymes, overload to renal function, anemia from erythrocyte destruction, and direct reaction, respectively, 4) there seems to occur a series of direct and adaptive reactions to exclude the "foreign compounds" which do not show any specific biological activities, 5) it is also considered that there is a possibility to establish a correlation between toxicological findings or target organs/tissues of both acute and subacute toxicities by their continuity. Therefore, in the present study, a predicting equation of subacute (28-day repeated dose) toxicity is attempted to develop from acute (single dose) toxicity data by considering both common mechanisms and biological factors for non-congeneric industrial chemical substances.

Semiautomatic mutual inductance bridge for magnetic susceptibility measurements.

A semiautomatic Hartshorn bridge has been designed to improve the complicated adjusting procedures of the bridge in the measurements of magnetic susceptibilities. The bridge is useful for a continuous recording of the susceptibility of a sample, especially when it varies with time.

New circuit for a constant voltage supply using series-shunt regulation.

A new circuit for constant voltage supply with series-shunt regulation has been designed to improve the suppression ratio of the incoming fluctuating noise. A typical example applicable to instruments with high sensitivity is also reported.

Temperature inhomogeneity in the glass mixing chamber of a (3)He/(4)He dilution refrigerator.

The temperature inhomogeneity in the glass mixing chamber of a (3)He/(4)He dilution refrigerator has been studied. The inhomogeneity is reduced to 10 mK by inserting a cylindrical spacer in the mixing chamber.

[MRI of lymphedema using short-TI-IR (STIR)].

Thirty four cases with lymphedema of the extremities were examined with MRI at 0.5 tesla. On T1-weighted image, the enlarged subcutaneous tissue and the subcutaneous trabecular structures were seen in all cases. Moreover, the trabecular structures in the enlarged subcutaneous tissue showed low signal intensity on T1-weighted image and high signal intensity on T2-weighted image in all cases. Additionally, in 12 of 15 cases examined by Short-TI-IR (STIR) image, the trabecular structures and fluid collections in the subcutaneous tissue were shown more definitely in high signal intensity than by T2-weighted image. We consider MRI using STIR is to be useful in the evaluation of edematous disease.