Lipid droplets in Arabidopsis thaliana leaves contain myosin-binding proteins and enzymes associated with furan-containing fatty acid biosynthesis.

Lipid droplets (LDs) are lipid storage organelles in plant leaves and seeds. Seed LD proteins are well known, and their functions in lipid metabolism have been characterized; however, many leaf LD proteins remain to be identified. We therefore isolated LDs from leaves of the leaf LD-overaccumulating mutant high sterol ester 1 (hise1) of Arabidopsis thaliana by centrifugation or co-immunoprecipitation. We then performed LD proteomics by mass spectrometry and identified 3,206 candidate leaf LD proteins. In this study, we selected 31 candidate proteins for transient expression assays using a construct encoding the candidate protein fused with green fluorescent protein (GFP). Fluorescence microscopy showed that MYOSIN BINDING PROTEIN14 (MYOB14) and two uncharacterized proteins localized to LDs labeled with the LD marker. Subcellular localization analysis of MYOB family members revealed that MYOB1, MYOB2, MYOB3, and MYOB5 localized to LDs. LDs moved along actin filaments together with the endoplasmic reticulum. Co-immunoprecipitation of myosin XIK with MYOB2-GFP or MYOB14-GFP suggested that LD-localized MYOBs are involved in association with the myosin XIK-LDs. The two uncharacterized proteins were highly similar to enzymes for furan fatty acid biosynthesis in the photosynthetic bacterium Cereibacter sphaeroides, suggesting a relationship between LDs and furan fatty acid biosynthesis. Our findings thus reveal potential molecular functions of LDs and provide a valuable resource for further studies of the leaf LD proteome.

Rubiscolin-6, a δ-Opioid Peptide from Spinach RuBisCO, Exerts Antidepressant-Like Effect in Restraint-Stressed Mice.

Rubiscolin-6 (Tyr-Pro-Leu-Asp-Leu-Phe) is produced by a pepsin digest of spinach d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and known to act as an agonist on δ-opioid receptor. Here, we showed that administration of rubiscolin-6 reduced immobility time in the tail suspension test in restraint-stressed mice without effect on locomotor activity. The antidepressant-like effect of rubiscolin-6 was blocked by a δ-opioid receptor antagonist, naltrindole. These results indicate that rubiscolin-6 exerts antidepressant-like effect through activation of δ-opioid receptor.

Human RGM249-derived small RNAs potentially regulate tumor malignancy.

The human noncoding RNA gene RGM249 has been shown to regulate the degree of cancer cell differentiation. In this study, we investigated the effects of 3 microRNA-like molecules digested from RGM249 on the loss of malignant properties in cancer cells in immunodeficient KSN/Slc mice. We utilized small interfering RNAs (siRNAs) alone or in combination with a cationized drug delivery system (DDS) consisting of atelocollagen or gelatin hydrogel microspheres. The results demonstrated growth inhibition and apoptosis and the inhibition of both neovascularization and metastasis, indicating that the DDSs effectively infiltrated the majority of tumor cells in vivo. Systemic administration of the 3 siRNAs inhibited the metastatic ability of malignant cells. Cotransfection of these siRNAs exerted a regulatory effect upon the genes involved in differentiation, pluripotency, and proliferation in cancer cells. These results suggest that RGM249-derived oligonucleotides may be involved in the regulation of metastasis, proliferation, and differentiation in vivo, and that the tested siRNAs may therefore represent a new anticancer therapeutic approach.

A noncoding RNA gene on chromosome 10p15.3 may function upstream of hTERT.

BACKGROUND: We attempted to clone candidate genes on 10p 14-15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid. RESULTS: RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells. CONCLUSION: These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT.

Clinical impact of serum transforming growth factor-alpha mRNA as a predictive biomarker for the prognosis of fulminant hepatitis.

PURPOSE: We previously reported that measuring serum telomerase reverse transcriptase (hTERT) mRNA with a quantitative, one-step, real-time RT-PCR was superior to conventional tumor markers for hepatocellular carcinoma and lung cancer. Here, we examined serum regeneration-related mRNA detection as a biomarker for fulminant hepatitis (FH). METHODS: In 53 patients, including 17 patients with acute hepatitis (AH), seven with severe hepatitis (SH), four with late-onset hepatic failure (LOHF), and 25 with FH, we measured serum mRNA levels of hTERT, hepatocyte growth factor (HGF), hepatocyte growth factor receptor (c-met), epidermal growth factor receptor (EGFR), and transforming growth factor-alpha (TGF-alpha). We examined the sensitivity and specificity of the technique in FH diagnosis as well as its clinical and prognostic significance compared with other clinical and prognostic tests. RESULTS: Serum copy number of TGF-alpha mRNA in FH on admission was significantly smaller than in AH and SH. In FH, TGF-alpha mRNA level was 10(6)-fold higher in survivors than in patients who died or received liver transplants (P = 0.034), although these patients were not discriminated by other clinical parameters. The sensitivity/specificity for prognosis in FH was 74.3/65.5% for TGF-alpha mRNA. Of four prognostic scoring systems, only logit-lambda was useful for prognosis assessment. CONCLUSIONS: TGF-alpha mRNA is an early predictor of FH outcome and a sensitive biomarker of lower regenerative liver capacity. This assay could help facilitate early therapy choice, such as liver transplantation.

A diagnostic evaluation of serum human telomerase reverse transcriptase mRNA as a novel tumor marker for gynecologic malignancies.

Human telomerase reverse transcriptase (hTERT) and epidermal growth factor receptor (EGFR) play an important role in many cancers including gynecological cancers. We previously reported the usefulness of a quantitative highly sensitive detection method for hTERT mRNA in the serum of cancer patients. By this method, we attempted to elucidate the diagnostic evaluation of serum hTERT mRNA for gynecologic malignancies. In 174 female patients with gynecological lesions (47 with ovarian lesions, 63 with uterine lesions, 2 with malignancies in other gynecological lesions, and 62 benign lesions) and 20 healthy individuals, we measured serum hTERT mRNA and EGFR mRNA by using the newly developed real-time quantitative RT-PCR. We examined their sensitivity and specificity in cancer diagnosis, clinical significance in comparison with conventional tumor markers, and their correlations with the clinical parameters by using multivariate analyses. Serum hTERT mRNA showed higher values in patients with gynecologic cancers than in those with benign diseases and healthy individuals. The hTERT mRNA level independently correlated with the presence of cancers (P=0.004 for both ovarian and uterine cancer) and clinical stage (P<0.001). The sensitivity and specificity of hTERT mRNA in cancer diagnosis was 74.4% and 74.1%, respectively. The hTERT mRNA level showed a significant correlation with CA125 by Pearson's relative test (P=0.035) and with histological findings in ovarian cancer by the Friedman test (P<0.004). EGFR mRNA did not display any differences between the diseases. hTERT mRNA is useful for diagnosing gynecologic cancer and is superior to conventional tumor markers. Therefore, serum hTERT mRNA is a novel and available biomarker for gynecologic malignancies.

Clinical usefulness of serum telomerase reverse transcriptase (hTERT) mRNA and epidermal growth factor receptor (EGFR) mRNA as a novel tumor marker for lung cancer.

Using a newly developed assay of telomerase reverse transcriptase (hTERT) mRNA in serum by real-time RT-PCR, we previously reported this assay to be superior to other tumor markers for hepatoma. In this study, we aimed to clarify its clinical significance as a biomarker for lung cancer. In 112 patients with lung tumor and 80 individuals without cancer, we measured serum hTERT mRNA and epidermal growth factor receptor (EGFR) mRNA levels, using a quantitative one-step real-time RT-PCR assay. We examined its sensitivity and specificity in lung cancer diagnosis, its clinical significance in comparison with other tumor markers, and its correlation with the clinical parameters using multivariate analyses and correlation relative tests. The copy number of serum hTERT mRNA was independently correlated with tumor size, tumor number, presence of metastasis and recurrence, and smoking (all P < 0.05). EGFR mRNA correlated with tumor number and clinical stage (both P < 0.05). The sensitivity and specificity in lung cancer diagnosis were 89.0% and 72.7% for hTERT mRNA, and 71.3% and 80.0% for EGFR mRNA, respectively. hTERT mRNA was superior to other tumor markers in lung cancer diagnosis. For both mRNAs, serum levels were significantly correlated with levels in lung cancer tissues (both P < 0.05). The copy number of hTERT mRNA significantly decreased after the surgical treatment. The data suggest that hTERT mRNA, especially when combined with EGFR mRNA, is a novel and excellent biomarker for pulmonary malignancies to diagnose and assess the clinical stage.

Serum human telomerase reverse transcriptase messenger RNA as a novel tumor marker for hepatocellular carcinoma.

PURPOSE: We previously reported the usefulness of a qualified highly sensitive detection method for human telomerase reverse transcriptase (hTERT) mRNA in serum with 89.7% sensitivity for hepatocellular carcinoma (HCC). In this study, we developed a quantitative detection method for serum hTERT mRNA and examined the clinical significance in HCC diagnosis. EXPERIMENTAL BACKGROUND: In 64 patients with HCC, 20 with liver cirrhosis, 20 with chronic hepatitis, and 50 healthy individuals, we measured serum hTERT mRNA by using the newly developed real-time quantitative reverse transcription-PCR with SYBR Green I. We examined its sensitivity and specificity in HCC diagnosis, clinical significance in comparison with other tumor markers, and its correlations with the clinical variables by using multivariate analyses. RESULTS: Serum hTERT mRNA showed higher values in patients with HCC than those with chronic liver diseases. hTERT mRNA expression was shown to be independently correlated with clinical variables such as tumor size, number, and degree of differentiation (P < 0.001, each). The sensitivity/specificity of hTERT mRNA and alpha-fetoprotein (AFP) mRNA in HCC diagnosis were 88.2%/70.0% for hTERT and 71.6%/67.5% for AFP, respectively. hTERT mRNA proved to be superior to AFP mRNA, AFP, and des-gamma-carboxy prothrombin in HCC diagnosis. Furthermore, hTERT mRNA in serum was associated with that in HCC tissue. CONCLUSIONS: The usefulness of hTERT mRNA expression in HCC diagnosis and its superiority to conventional tumor markers were shown. Therefore, serum hTERT mRNA is a novel and available marker for HCC diagnosis.