The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development

The products of Hox genes function in assigning positional identity along the anterior–posterior body axis during animal development. In mouse embryos, Hox genes located at the 3′ end of HoxA and HoxB complexes are expressed in nested patterns in the progenitors of the secondary heart field during early cardiogenesis and the combined activities of both of these clusters are required for proper looping of the heart. Using Hox bacterial artificial chromosomes (BACs), transposon reporters, and transgenic analyses in mice, we present the identification of several novel enhancers flanking the HoxB complex which can work over a long range to mediate dynamic reporter expression in the endoderm and embryonic heart during development. These enhancers respond to exogenously added retinoic acid and we have identified two retinoic acid response elements (RAREs) within these control modules that play a role in potentiating their regulatory activity. Deletion analysis in HoxB BAC reporters reveals that these control modules, spread throughout the flanking intergenic region, have regulatory activities that overlap with other local enhancers. This suggests that they function as shadow enhancers to modulate the expression of genes from the HoxB complex during cardiac development. Regulatory analysis of the HoxA complex reveals that it also has enhancers in the 3′ flanking region which contain RAREs and have the potential to modulate expression in endoderm and heart tissues. Together, the similarities in their location, enhancer output, and dependence on retinoid signaling suggest that a conserved cis-regulatory cassette located in the 3′ proximal regions adjacent to the HoxA and HoxB complexes evolved to modulate Hox gene expression during mammalian cardiac and endoderm development. This suggests a common regulatory mechanism, whereby the conserved control modules act over a long range on multiple Hox genes to generate nested patterns of HoxA and HoxB expression during cardiogenesis.     

Expression of the Cellulase (FI-CMCase) Gene of Aspergillus aculeatus in Escherichia coli

FI-CMCase cDNA of Aspergillus aculeatus was expressed in Escherichia coli by using the tac promoter of E. coli. Transformants of E. coli harboring a plasmid pHEM06 containing mature form FI-CMCase cDNA produced FI-CMCase in the cytoplasm of the cells. The enzyme from E. coli cells was purified to yield 56% and it was immunological identical to that of FI-CMCase purified from A. aculeatus.     

FEEDING ENTRAINMENT OF DAILY RHYTHMS OF LOCOMOTOR ACTIVITY AND CLOCK GENE EXPRESSION IN ZEBRAFISH BRAIN

Light and feeding cycles strongly synchronize daily rhythms in animals, which may, as a consequence, develop food anticipatory activity (FAA). However, the light/food entraining mechanisms of the central circadian oscillator remain unknown. In this study, we investigate the existence of FAA in seven groups of zebrafish subjected to a light/dark (LD) cycle or constant light (LL) and different feeding regimes (random, fasting, and feeding in the middle of the light phase or dark phase). The aim was to ascertain whether the daily rhythm of behavior and clock gene (per1 and cry1) expression in the zebrafish brain was entrained by the light and feeding regime. The results revealed that FAA developed in zebrafish fed daily at a fixed time, under LD and under LL. Zebrafish displayed locomotor activity mostly during the daytime, although the percentage of activity during the light phase varied depending on feeding time (ranging from 93.2% to 63.1% in the mid-light and mid-dark fed groups, respectively). However, the different feeding regimes failed to modify the daily rhythm of per1 and cry1 expression in the zebrafish brain under LD (approximate acrophases [peak times] at ZT22 and ZT4, respectively; lights-on =?ZT0). Under LL, per1 and cry1 expression did not show significant daily rhythmicity, regardless of the feeding regime. These findings indicate that, although schedule-fed zebrafish developed FAA as regards locomotor activity, feeding had little effect on clock gene expression in whole brain homogenates, suggesting the feeding-entrainable oscillator may be located elsewhere or at specific brain sites. (Author correspondence: jasanchez@um.es)     

Clock Gene Expression Levels and Relationship With Clinical and Pathological Features in Colorectal Cancer Patients

The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1Ε expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p?=?.002), PER1 (p?=?.002), PER2 (p?=?.011), PER3 (p?=?.003), and CRY2 (p?=?.012) were lower, whereas the expression level of TIM (p?=?.044) was higher. No significant difference was observed in the expression levels of CLOCK (p?=?.778), CRY1 (p?=?.600), CSNK1Ε (p?=?.903), and TIPIN (p?=?.136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p?=?.026), and female sex (p?=?.005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p?=?.015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p?= .013) and associated with TNM stages III–IV (p?=?.005) and microsatellite instability (p?=?.015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p?=?.010), PER3 (p?= .010), and CSNKIE (p?=?.024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions. (Author correspondence: g.mazzoccoli@tin.it)     

ASSOCIATIONS OF METABOLIC PARAMETERS AND ETHANOL CONSUMPTION WITH MESSENGER RNA EXPRESSION OF CLOCK GENES IN HEALTHY MEN

Recent studies suggest that the impairment of circadian clock function causes various pathological conditions, such as obesity, diabetes, and alcoholism, and an altered mRNA expression of clock genes was found under these conditions. However, it remains to be determined whether clock gene expression varies depending on metabolic conditions even in healthy people. To address this issue, we investigated the associations of metabolic parameters and alcohol consumption with mRNA expression of clock genes (CLOCK, BMAL1, PER1, PER2, and PER3) in peripheral blood cells obtained from 29 healthy non-obese elderly men (age 51–78 yrs) who adhered to a regular sleep-wake routine, through a single time-of-day venous blood sampling at ～09:00?h. There were significant correlations between (1) waist circumference and mRNA level of PER1 (r?=?0.43), (2) plasma glucose concentration and PER2 (r?=?0.50), (3) ethanol consumption and BMAL1 (r?=?0.43), and (4) serum γ-GTP concentration (a sensitive marker of alcohol consumption) and PER2 (r?=?0.40). These results suggest mRNA expression of clock genes is associated with obesity, glucose tolerance, and ethanol consumption even in healthy people. (Author correspondence: akiofuji@jichi.ac.jp)     

Organization,development, and effects of infraorbital nerve transection on galanin binding sites in the trigeminal brainstem complex

Previous experiments from this laboratory have indicated that transection of the infraorbital nerve (ION, the trigeminal \[V] branch that supplies the mystacial vibrissae follicles) at birth and in adulthood has markedly different effects on galanin immunoreactivity in the V brainstem complex. Adult nerve transection increases galanin immunoreactivity in the superficial layers of V subnucleus caudalis (SpC) only, while neonatal nerve transection results in increased galanin expression in vibrissae-related primary afferents throughout the V brainstem complex. The present study describes the distribution of binding sites for this peptide in the mature and developing V ganglion and brainstem complex and determines the effects of neonatal and adult ION damage and the associated changes in galanin levels upon their distribution and density. Galanin binding sites are densely distributed in all V brainstem subnuclei and are particularly dense in V subnucleus interpolaris and the superficial layers of SpC. They are present at birth (P-0) and their distribution is similar to that in adult animals. Transection of the ION in adulthood and examination of brainstem 7 days later indicated marked reductions in the density of galanin binding sites in the V brainstem complex. With the exception of the superficial laminae of SpC, the same reduction in density remained apparent in rats that survived 45 days after nerve cuts. Transection of the ION on P-0 resulted in no change in the density of galanin binding sites in the brainstem after either 7 or 60 days survival. These results indicate that densely distributed galanin binding sites are present in the V brainstem complex of both neonatal and adult rats, that they are located in regions not innervated by galanin-positive axons, and that their density is not significantly influenced by large lesion-induced changes in the primary afferent content of their natural ligand.     

Spatial summation of heat induced pain within and between dermatomes

The aim was to study spatial summation within and between ipsi- and contralateral dermatomes at different painful temperatures. For heat stimulation we used a computer controlled thermofoil based thermode. The thermode area could be varied in five discrete steps from 3.14 to 15.70 cm2. When we applied the stimuli within a dermatome, the mean heat pain threshold decreased significantly from 45.6 to 43.5 C as the area was increased from minimum (3.14 cm2) to maximum (15.70 cm2). When the areas were increased involving different dermatomes (both ipsi- or contralateral), we found similar decreases in pain threshold. Spatial summation was also found within and between dermatomes at supra-threshold temperatures (46, 48, 50 C).The study shows that spatial summation of pain is most likely a mechanism acting across segments and is existing from pain threshold to tolerance.     

Effects of Colonization of a Bacterial Endophyte,Azospirillum sp. B510, on Disease Resistance in Rice

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, neither salicylic acid (SA) accumulation nor expression of pathogenesis-related (PR) genes was induced by interaction with this bacterium, except for slight induction of PBZ1. These results indicate the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.