Microarray analysis of <Emphasis Type="Italic">Arabidopsis</Emphasis> plants in response to allelochemical <Emphasis Type="SmallCaps">l</Emphasis>-DOPA

Velvetbean (Mucuna pruriens) plants impede the growth of neighboring plants. One compound, 3-(3′,4′-dihydroxyphenyl)-l-alanine (l-DOPA), is responsible for the allelopathic capacity of velvetbean. This compound is an active allelochemical that decreases root growth of several plant species. In mammals, l-DOPA is a well-known therapeutic agent for the symptomatic relief of Parkinson’s disease. However, its mode of action in plants is still not well understood. To address such issues, gene expression in Arabidopsis thaliana plants, which had been exposed to l-DOPA, was analyzed using DNA microarrays. After 6 h of l-DOPA exposure, the expression of 110 genes was significantly upregulated, and the expression of 69 genes was significantly downregulated. These induced genes can be divided into different functional categories, mainly on the basis of subcellular localization, metabolism, and proteins with a binding function or cofactor requirement. Based on these results, we suggest that l-DOPA acts by two mechanisms: it influences amino acid metabolism and deregulates metal homeostasis, especially that of iron, which is required for the fundamental biological processes of all organisms.     

Functional importance of inositol-1,4,5-triphosphate-induced intracellular Ca2+ mobilization in galanin-induced microglial migration

Galanin (GAL) is a neuropeptide which is up-regulated following neuronal axotomy or inflammation. One subtype of GAL receptor (GalR2) is reported to be expressed in the brain's immune cell population, microglia. In the present study, we investigated the effect of GAL on microglial migration and compared the mechanism with that of bradykinin (BK). GAL significantly increased the migration of rat cultured microglia at 0.1 pM. The GAL-induced signal cascade was partly similar to that induced by BK. It was not dependent on G(i/o) protein but involved activation of protein kinase C, phosphoinositide 3-kinase and Ca(2+)-dependent K(+) channels. However, reverse-mode activation of the Na(+) /Ca(2+) -exchanger 1 was not involved in GAL-induced microglial migration, unlike BK-induced migration. Likewise, nominally-free extracellular Ca(2+) inhibited BK-induced migration but not GAL-induced migration. An inositol-1,4,5-triphosphate receptor antagonist significantly inhibited GAL-induced migration. GAL-induced Ca(2+) signaling did not induce nitric oxide synthase expression, but up-regulated class II major histocompatibility complex expression. These results indicate that activation of inositol-1,4,5-triphosphate receptor and increase in intracellular Ca(2+) are important for GAL-induced migration and immunoreactivity in microglia. The differences in down-stream signal transduction induced by GAL and BK suggest that GAL and BK may control distinct microglial functions under pathological conditions.     

Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9–Rad1–Hus1 complex (9–1–1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9–1–1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.     

NCYM,a Cis-Antisense Gene of MYCN,Encodes a De Novo Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.     

Associations between renal impairment and anemia in older,rural Japanese men: the Nagasaki Island study

Background

Renal impairment is known to be associated with atherosclerosis, which in turn is reported to be positively associated with hemoglobin levels. In addition, renal impairment is known to be associated with a form of anemia known as renal anemia.

Methods

To clarify the associations between renal impairment and anemia, we conducted a cross-sectional study of 1,105 60 to 89-year-old men, who were not taking medication for anemia and were undergoing general health check-ups.

Results

Compared with non-chronic kidney disease, chronic kidney disease (CKD) with a glomerular filtration rate (GFR) <60 mL/min/1.73 m² was found to constitute a significant risk of anemia. However, we noted that this risk was lower for mild renal impairment (60 mL/min/1.73 m² ≤ GFR <90 mL/min/1.73 m²). Compared with the non-CKD reference group, the classical cardiovascular risk factors adjusted odds ratio (OR) for anemia was 1.81 (1.23 to 2.68) and compared with the normal renal function (GFR ≥90 mL/min/1.73 m²) reference group, the ORs for mild renal impairment and CKD were 0.26 (0.15 to 0.47) and 0.60 (0.33 to 1.09).

Conclusions

Independent from classical cardiovascular risk factors, CKD, which was identified during general health check-ups, appeared to constitute a significant risk of anemia for older Japanese men. For mild renal impairment, however, this association was a reduced risk of anemia and thus possibly a higher risk of atherosclerosis.     

Biochemical and genetic heterogeneity of staphylococcal protein A

Abstract We investigated the biochemical and genetic heterogeneity of protein A from Staphylococcus aureus . SpA genes ( spas ) of various strains were heterogeneous when detected as Dra I and Eco RV fragments of chromosomal DNA. Polymerase chain reaction using primers to detect DNA encoding the IgG-binding domains in spa revealed that they numbered between 2 and 5. Protein A from several S. aureus strains showed two types of reactivities to immunoglobulins in normal canine serum according to the number of active domains.     

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples.     

Perspective: functional genomics towards new biotechnology in medicinal plants

The secret of chemical diversity and function of specialized metabolites in medicinal plants will be unveiled by study of functional genomics at an unprecedentedly rapid rate in the coming years. This is mostly ascribed to the remarkable advancement in the high-throughput DNA sequencing together with other omics technologies such as metabolomics, in particular, due to drastic reduction in the cost of acquiring, storing and analyzing massive omics datasets. Once the genes involved in a biosynthetic pathway of specialized compounds in plants are elucidated, synthetic biology or genome editing can be applied to produce the target compounds in an engineered organism or to manipulate the pathway in planta. Coupled with these advancements in pathway elucidation approaches, modern plant biotechnology strategies are bound to significantly contribute to the sustainable development goals set by United Nations.     

Expression of gibberellin 3 beta-hydroxylase gene in a gravi-response mutant, weeping Japanese flowering cherry.

Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.