Histone Demethylases JMJ30 and JMJ32 Modulate the Speed of Vernalization Through the Activation of FLOWERING LOCUS C in Arabidopsis thaliana.

Vernalization is the promotion of flowering after prolonged exposure to cold. In Arabidopsis thaliana, vernalization induces epigenetic silencing of the floral repressor gene FLOWERING LOCUS C (FLC). Among the repressive epigenetic marks, the trimethylation of lysine 27 on histone H3 proteins (H3K27me3) is a critical contributor to the epigenetic silencing of FLC. The deposition of H3K27me3 is mediated by Polycomb Repressive Complex 2 (PRC2). Conversely, the elimination of H3K27me3 is mediated by histone demethylases, Jumonji-C domain-containing protein JMJ30 and its homolog JMJ32. However, the role of JMJ30 and JMJ32 in vernalization is largely unknown. In this study, we found that cold treatment dramatically reduced the expression levels of JMJ30 and did not reduce those of JMJ32. Next, by using the genetic approach, we found that the flowering of jmj30 jmj32 was accelerated under moderate vernalized conditions. Under moderate vernalized conditions, the silencing of FLC occurred more quickly in jmj30 jmj32 than in the wild type. These results suggested that the histone demethylases JMJ30 and JMJ32 brake vernalization through the activation of FLC. Our study suggested that PRC2 and Jumonji histone demethylases act in an opposing manner to regulate flowering time via epigenetic modifications.

Transluminal Approach with Bubble-Seeded Histotripsy for Cancer Treatment with Ultrasonic Mechanical Effects.

Bubble-seeded histotripsy (BSH) is a newly developed ultrasound-based mechanical fractionation technique using locally injected phase change nanodroplets (PCNDs) as sensitizers. The PCNDs are a kind of microbubble precursor compressed into submicron-size in droplets form, which were designed for local administration and will expand into microbubbles under ultrasound exposure. Previously, we reported that a combination of PCNDs injection and pulsed high-intensity focused ultrasound (pHIFU) with an acoustic intensity as low as about 3 kW/cm2 at 1.1 MHz, which is similar to the acoustic intensity of currently available HIFU coagulation therapy, was enough to induce tissue fractionation after significant antitumor effects in an in vivo study. Toward therapeutic application of BSH to deep-seated tissues such as the pancreas, the transluminal approach, using endoscopic ultrasound was thought to be ideal. Therefore, for a preliminary examination, we developed a new transducer with a small aperture (20- × 20-mm square) and long focal length (35 mm), operating at 2.1 MHz that could be attached to an EUS-mimicking probe. With the newly developed transducer and locally injected PCNDs, predictable tissue mechanical fractionation was observed in both ex vivo and in vivo studies at acoustic intensities that were too low to induce any significant bioeffects (around 4 kW/cm2) without using PCNDs. For in situ monitoring of the treatment site during a procedure, the degree of attenuation of microbubble motions after exposing the microbubbles to pHIFU was monitored, using ultrafast echographic imaging. Microbubble movements were observed to be largest at 25-30 s after pHIFU exposure. On the contrary, after 40 s, the movement of microbubbles decreased to the same level as at the start of the procedure, suggesting that an overdose of pHIFU exposure causes coagulation attributable to the thermal effect caused by absorption of the energy. Those results were promising for expanding the application of BSH for a transluminal approach, using a small transducer under real-time monitoring.

New approach for local cancer treatment using pulsed high-intensity focused ultrasound and phase-change nanodroplets.

PURPOSE: The aim of this study was to investigate the combination effects of pulsed HIFU (pHIFU) and phase-change nanodroplets (PCND) as a sensitizer on efficient induction of mechanical effects of pHIFU and chemically enhanced tumor growth inhibition for local anti-tumor therapy. METHOD: Changes in growth of colon 26 tumor tissue inoculated onto CDF1 mice were evaluated by the following treatments. (1) pHIFU exposure (1.1 MHz, 3.2 kW/cm(2), 300 cycles, and 50 ms interval) for 60 s, (2) PCND (1 %) injection, (3) adriamycin (4 mg/kg) injection, (4) pHIFU exposure after PCND injection, and (5) pHIFU exposure after PCND + adriamycin injection simultaneously. RESULTS: Significant changes in tumor growth were observed in the group with combination of pHIFU and PCND, although single therapy did not show any significant difference. PCND enhanced mechanical tissue fractionation by pHIFU, which was detectable by Real-time tissue elastography. Moreover, the combination of pHIFU and PCND + Adriamycin suppressed the tumor growth for 2 weeks, and 3 of 4 mice did not show any sign of regrowth during the 30-day observation. CONCLUSION: The combination of pHIFU and PCND exerted a significant anti-tumor effect and may be a new candidate for treatment of locally advanced cancer.

Analysis of the anatomic subsites, gender and age in unresectable advanced colorectal carcinomas in Tochigi, Japan suggests a shift in location towards the right side colon in elderly patients treated with cetuximab.

Colorectal cancer is a frequently occurring cancer whose incidence has shown a marked increase in recent years. Additionally, an increase in right side colon in elderly patients has been identified. Therefore, a clinicopathological study was conducted in 49 patients with unresectable advanced colorectal carcinomas to elucidate the association of clinicopathological characteristics and K-ras mutation. Of the 49 patients included in this study, 24 were aged <60 years with a male/female (M/F) ratio of 16/8 and 25 patients were aged ≥60 years with a M/F ratio of 16/9. Of the patients aged ≥65 years, 15 patients were enrolled as controls and the M/F ratio was 9/6. Results revealed that with regard to the subsite of cancer, unresectable advanced colorectal carcinomas developed in the right-sided colon in 13 patients, left-sided colon in 19 patients and rectum in 17 patients. Right-sided colon carcinomas were commonly identified in the elderly patients aged ≥65 years, with a marked tendency in the female patients (P=0.024). Immunostaining was performed for the epidermal growth factor receptor (EGFR) antibody in 40 patients to determine whether the K-ras gene would yield positive results. The mutant K-ras gene was identified in 8 patients (20%) and the frequency was lower compared with that of the normal colorectal carcinomas. Anti-EGFR antibody (cetuximab) is considered to be a molecularly targeted agent for unresectable advanced colorectal carcinomas. The increase in incidence of right-sided colon carcinomas as well as the increase in the number of patients presenting with colorectal carcinomas means this issue should be addressed. Sessile serrated adenoma/polyp (SSA/P) with b-raf mutation and CIMP (CpG island methylator phenotype) abnormality as a precursor lesion of right-sided colon carcinoma is common and since cetuximab refractory wild-type K-ras/mutant b-raf colorectal carcinoma may increase in elderly patients and patients with right-sided colon carcinoma, a simultaneous examination for the K-ras and b-raf gene abnormalities for the treatment of colorectal cancer using anti-EGFR antibody (cetuximab) is crucial. In addition, the multidisciplinary assessments regarding the effect of such treatments is likely to be determined based on cumulative results, such as the duration of patient survival.

Maternal enrichment affects prenatal hippocampal proliferation and open-field behaviors in female offspring mice.

The maternal environment is thought to be important for fetal brain development. However, the effects of maternal environment are not fully understood. Here, we investigated whether enrichment of the maternal environment can influence prenatal brain development and postnatal behaviors in mice. An enriched environment is a housing condition with several objects such as a running wheel, tube and ladder, which are thought to increase sensory, cognitive and motor stimulation in rodents compared with standard housing conditions. First, we measured the number of BrdU-positive cells in the hippocampal dentate gyrus of fetuses from pregnant dams housed in an enriched environment. Our results revealed that maternal enrichment influences cell proliferation in the hippocampus of female, but not male, fetuses. Second, we used the open-field test to investigate postnatal behaviors in the offspring of dams housed in the enriched environment during pregnancy. We found that maternal enrichment significantly affects the locomotor activity and time spent in the center of the open-field in female, but not male, offspring. These results indicate that maternal enrichment influences prenatal brain development and postnatal behaviors in female offspring.

Ghrelin alters postnatal endocrine secretion and behavior in mouse offspring.

Maternal bioactive substances, such as hormones and neuropeptides, are thought to be essential for fetal development. Recently, ghrelin, a gastrointestinal peptide, has been shown to pass through the rat placenta. The ghrelin receptor, growth hormone secretagogue receptor (GHS-R), has been shown to be expressed in the rat fetal central nervous system, and plasma ghrelin levels are related to birth weight in the rodent and human. In the present study, we report a role of maternal ghrelin in mouse fetal brain development. When ghrelin was administrated to pregnant mice, pups exhibited suppression of exploratory behavior in an open-field (OF) test. Control pups, however, remained for longer periods of time in the center area, correlating with exploratory behavior. Basal corticotropin-releasing hormone (CRH) plasma levels were greater in pups from ghrelin-treated dams, and did not change in response to acute restraint stress. Moreover, reduced growth hormone secretagogue receptor and neuropeptide Y mRNA expression was observed in the hypothalamus at postnatal day 3 and remained until 16 weeks of age. In addition, under physiological condition, increased maternal ghrelin plasma levels following repeated restraint stress to the dam had effect on the increase in fetal plasma acyl ghrelin levels. These results suggest that maternal ghrelin affect fetal plasma ghrelin levels and alters endocrine systems and behaviors of offspring.

Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway.

The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and STE11. These mutants were characterized using an HOG-dependent reporter gene, 8xCRE-lacZ. We found that Cdc42, in addition to binding and activating the PAK-like kinases Ste20 and Cla4, binds to the Ste11-Ste50 complex to bring activated Ste20/Cla4 to their substrate Ste11. Activated Ste11 and its HOG pathway-specific substrate, Pbs2, are brought together by Sho1; the Ste11-Ste50 complex binds to the cytoplasmic domain of Sho1, to which Pbs2 also binds. Thus, Cdc42, Ste50, and Sho1 act as adaptor proteins that control the flow of the osmostress signal from Ste20/Cla4 to Ste11, then to Pbs2.

Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1.

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548). In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.