A distinct function of the retinoblastoma protein in the control of lipid composition identified by lipidomic profiling.

Here, by combining lipidomics with transcriptome analysis, we demonstrate that Rb depletion in mouse embryonic fibroblastss induces significant alterations in their lipid composition. We discovered that Rb depletion induced increase in lysophosphatidylserine, diacylglycerol (DAG), fatty acid (FA), acylcarnitine, phosphatidylcholine (PC), arachidonoyl ethanolamine, and decrease in phosphatidylglycerol, monoacylglycerol, without change in total lipid per protein levels. Analysis of the acyl chain composition of DAG, PC and phosphatidylserine revealed increase of saturated and mono-unsaturated acyl chains with specific carbon chain length. Consistently, we observed that Rb depletion increased the levels of fatty acids with the corresponding carbon chain length and number of carbon-carbon double bondssuch as myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0) and all forms of FA 18:1. Microarray analysis revealed that Rb depletion induced significant upregulation of enzymes involved in elongation and desaturation of fatty acids. Among these, we found that elongation of long chain fatty acid family member 6 (Elovl6) and stearoyl-CoA desaturase 1 (Scd1) are the most robustly controlled by Rb possibly through E2F and sterol regulatory element-binding protein transcription factors. Depletion of Elovl6 or Scd1 significantly suppressed colony formation, sphere formation and xenograft tumor growth of Rb-deficient tumor cells. Suppression of self-renewal by the SCD1 inhibitor was rescued upon supplementation of the mono-unsaturated fatty acids generated by this enzyme. This study suggests a novel role for Rb in suppressing the malignant progression of tumors by controlling the lipid composition.

The RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity.

Retinoblastoma (RB) protein inactivation during tumor progression is often associated with acquisition of immature phenotypes and resistance to therapy. Determination of an RB inactivation signature in a context of gaining undifferentiated phenotype in a p53-null sarcoma system revealed a critical role for interleukin (IL)-6. Using a Gene Set Enrichment Analysis (GSEA), we discovered that poorly differentiated breast cancers are enriched for this RB inactivation signature. Accelerated IL-6 secretion following RB inactivation in an RB-intact luminal-type breast cancer cell line MCF-7 promoted a positive feed forward loop between IL-6 and STAT3 driving tumor growth and endocrine therapy resistance. In addition, some of RB-intact basal-like type breast cancer cell lines exhibited a similar phenotype following RB depletion. The mechanism whereby RB inactivation increases IL-6 production in MCF-7 cells appeared to involve fatty acid oxidation (FAO)-dependent mitochondrial metabolism and c-Jun NH(2)-terminal kinase (JNK). In addition, IL-6, via STAT3-mediated feedback to mitochondria, autonomously adjusts mitochondrial superoxide to levels suitable to maintain stem cell-like activity. The gene expression profile of luminal-type breast cancer patients with low RB expression revealed high enrichment of genes involved in mitochondrial respiration and downstream targets of IL-6. These findings unveiled an unexpected strategy whereby RB suppresses malignant features of cancer cells through metabolic reprogramming and cell-autonomous inflammation.

Simultaneous topographic and fluorescence imagings of recombinant bacterial cells containing a green fluorescent protein gene detected by a scanning near-field optical/atomic force microscope.

A scanning near-field optical/atomic force microscope (SNOAM) system was applied for simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever and as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of GFP, since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.

[Dental health examination in infancy and early childhood. Part 1. The results of questionnaires].

The purpose of this study was to find the characteristic nursing environments of children in order to confirm the methods of the dental health examination in infancy and early childhood. So questionnaires were performed. The subjects were 436 children, 218 boys and 218 girls, who had undergone successively both dental healthy examinations at the age of 1.5 and 3 years. The following results were obtained: 1. Concerning occupation, 63% of their fathers were office workers, and 75% of their mothers had no occupation. More than 90% of parents were both at the age of 20's or 30's. 2. Eighty percent of the children was nursed by their mothers in the daytime at the age of both 1.5 years and 3.5 years. 3. Abnormalities during the gestational period were present in about 1/3 pregnant women, but were mild in most cases. About 20% had abnormalities at the time of deliver; however, less than 5% showed abnormal courses thereafter. The mean birth weight was 3,104.6 g, which is lower than that in Japan. 4. Breast feeding was most highly observed at the age of 3 months; 80% took baby food at the age of 6 months; milk intake was markedly increased at the age of 12 months. 5. Concerning kinds of all between-meal snacks, sweet snacks were slightly increased, showing more variety in food. However, a consciousness of oral hygiene in parents seemed to show a favorable tendency on the data on regularity, number and place of eating snacks. 6. Although a majority of the children used a toothbrush after the age of 1.5 years, 50% and 25% of the children did not brush teeth every day at the age of 1.5 years and 3.5 years, respectively. Therefore the author recognized the necessity of health guidance and aftercare.

[Dental health examination in infancy and early childhood. Part 2. The results of dental survey and screening of dental caries].

The purpose of this study was to analyze the relationship between the caries incidence, oral environment at the age of 1.5 years and the prevalence of the caries at the age of 3.5 years in order to establish the methods of dental health surveys on infants and to prevent dental caries. Oral health examination and caries activity (Cariostat) test were performed on 436 children successively undergoing dental health examinations of 1.5-year-old and 3-year-old children, and the following results were obtained: 1. Concerning the condition of dental caries prevalence, percentages of children with dental caries, mean deft and mean defs were 10.6%, 0.36 and 0.68 at the age of 1.5 years and 67.2% 4.43 and 8.41 at the age of 3.5 years, respectively. 2. According to dental surfaces, the mesial proximal surface and lingual surface of primary central incisor in the upper jaw showed a high defs rate of 4.6% at the age of 1.5 years. At the age of 3.5 years, the occlusal surface of primary second molar in the lower jaw, the mesial proximal surface of primary central incisor in the upper jaw and the occlusal surface of primary second molar in the upper jaw had defs rates as high as over 35%. 3. Concerning the condition of occlusion, normal occlusions and deep overbites appeared at almost the same frequencies occupying about 70% of overall occlusions. 4. A risk of dental caries became higher when the body weight at birth was higher, eruption occurred earlier, a fissure in the enamel of primary was deeper, Cariostat pH was lower, and/or no interdental spaces between primary incisors in the upper jaw; therefore the author recognized the necessity of individual health guidance and aftercare.