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The aim of this study was to clarify the structure and regeneration status of mixed beech (Fagus crenata) forest on Mt. Toishi in the Sangun mountain range in northern Kyushu, a relatively low‐latitude region in Japan, with emphasis on forests at lower elevations. We focused on a mixed beech forest at a low elevation of Mt. Toishi and compared the forest structure of the target stands, including tree species and size, with the forest structure near the peaks of Mt. Toishi and Mt. Sangun. Deciduous broad‐leaf trees were dominant near the peaks, while evergreen broad‐leaf trees dominated at lower elevations, whereas the relative dominance of beech trees was not significantly different. Near the peaks, the number of large‐diameter beech trees forming the canopy decreased, and small‐diameter trees were present. The proportion of small‐sized trees was lower in the low‐elevation forest area than in the forest area near the peak. Even at lower elevations, juvenile beech trees grew in limited areas on steep slopes along forest roads. These sites suggested that regeneration of beech populations is possible even at low elevation. [ABSTRACT FROM AUTHOR]

Introduction Most human cancers originate from epithelial cells. They then progress through a series of genetic alterations and expand their territory. In the case of colorectal cancers, one of the [...], Intracellular [Mg.sup.2+] levels are strictly regulated; however, the biological importance of intracellular [Mg.sup.2+] levels and the pathways that regulate them remain poorly understood. Here, we determined that intracellular [Mg.sup.2+] is important in regulating both energy metabolism and tumor progression. We determined that CNNM4, a membrane protein that stimulates [Mg.sup.2+] efflux, binds phosphatase of regenerating liver (PRL), which is frequently overexpressed in malignant human cancers. Biochemical analyses of cultured cells revealed that PRL prevents CNNM4-dependent [Mg.sup.2+] efflux and that regulation of intracellular [Mg.sup.2+] levels by PRL and CNNM4 is linked to energy metabolism and AMPK/mTOR signaling. Indeed, treatment with the clinically available mTOR inhibitor rapamycin suppressed the growth of cancer cells in which PRL was overexpressed. In [Apc.sup.Δ14/+] mice, which spontaneously form benign polyps in the intestine, deletion of Cnnm4 promoted malignant progression of intestinal polyps to adenocarcinomas. IHC analyses of tissues from patients with colon cancer demonstrated an inverse relationship between CNNM4 expression and colon cancer malignancy. Together, these results indicate that CNNM4-dependent [Mg.sup.2+] efflux suppresses tumor progression by regulating energy metabolism.

Phosphatase of regenerating liver (PRL) is a family of protein tyrosine phosphatases (PTPs) that are anchored to the plasma membrane by prenylation. They are frequently overexpressed in various types of malignant cancers and their roles in cancer progression have received considerable attention. Mutational analyses of PRLs have shown that their intrinsic phosphatase activity is dispensable for tumor formation induced by PRL overexpression in a lung metastasis model using melanoma cells. Instead, PRLs directly bind to cyclin M (CNNM) Mg2+ exporters in the plasma membrane and potently inhibit their Mg2+ export activity, resulting in an increase in intracellular Mg2+ levels. Experiments using mammalian culture cells, mice, and C. elegans have collectively revealed that dysregulation of Mg2+ levels severely affects ATP and reactive oxygen species (ROS) levels as well as the function of Ca2+‐permeable channels. Moreover, PRL overexpression altered the optimal pH for cell proliferation from normal 7.5 to acidic 6.5, which is typically observed in malignant tumors. Here, we review the phosphatase‐independent biological functions of PRLs, focusing on their interactions with CNNM Mg2+ exporters in cancer progression. [ABSTRACT FROM AUTHOR]

Aims: Mg2+ is fundamental for life, and its shortage severely impairs vital functions. However, whether excessive Mg2+ has beneficial or adverse effects has remained unknown. To clarify this issue, we analyzed the effect of suppressing the functions of Cyclin M (CNNM) Mg2+ efflux transporters in various experimental systems. Results: Investigation of short-lived Caenorhabditis elegans worms mutated for CNNM genes revealed reactive oxygen species (ROS) augmentation in intestinal cells, coincidently with high levels of Mg2+. Knockdown of gtl-1, encoding Mg2+-incorporating channel into intestinal cells, reduced ROS levels and restored life span, confirming the causative role of excessive Mg2+. Also, inactivation of orthologous CNNM in human cultured cells and mice by RNA interference, expression of CNNM-inhibiting protein, phosphatase of regenerating liver 3, or gene knockout resulted in ROS overproduction. Moreover, biochemical analyses revealed that excessive Mg2+ stimulates adenosine triphosphate overproduction and accelerates mitochondrial electron transport, whose suppression shut down ROS generation. Innovation and Conclusion: These results provide definitive evidence that excessive Mg2+ drives overproduction of ROS by affecting energy metabolism, implying the crucial importance of the tight regulation of intracellular Mg2+ levels. [ABSTRACT FROM AUTHOR]

Cyclin M (CNNM) family proteins are evolutionarily conserved Mg2+ transporters. They extrude Mg2+ from cells and maintain intracellular Mg2+ levels within the normal range. Moreover, they play an important role in Mg2+ (re)absorption in the intestine and kidney by mediating the directional transport of Mg2+ across epithelial tissue from the tubular lumen to the body inside. Mg2+ efflux is suppressed by the direct binding with phosphatase of regenerating liver (PRL), and the formation of the complex is dynamically regulated by cysteine phosphorylation of PRL. The dysfunction of CNNM family proteins is responsible for inherited hypomagnesemia, as well as various intractable diseases, such as cancer and hypertension. Through multiple functional analyses of CNNM family proteins, the biomedical significance of the proper regulation of Mg2+ levels has been elucidated. [ABSTRACT FROM AUTHOR]

Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK. [ABSTRACT FROM AUTHOR]

Transcellular Mg2+ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg2+ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg2+ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg2+ by exchanging intracellular Mg2+ with extracellular Na+. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg2+ extrusion activity. These results demonstrate the crucial importance of Mg2+ extrusion by CNNM4 in organismal and topical regulation of magnesium. [ABSTRACT FROM AUTHOR]

Numerous studies indicate that reactive oxygen species (ROS) are not merely cellular by-products of respiration, but are able to modulate various signalling pathways and play certain physiological roles. Recent studies have revealed the importance of translating ROS-generation to activation/suppression of specific signalling pathways. The Wnt signalling pathway, which is essential for early development and stem cell maintenance, is also regulated by ROS. A thioredoxin-related protein, nucleoredoxin (NRX), governs ROS-stimulated Wnt signalling in a temporal manner. NRX usually interacts with Dishevelled (Dvl), an essential adaptor protein for Wnt signalling, and blocks the activation of the Wnt pathway. Oxidative stress causes dissociation of NRX from Dvl, which enables Dvl to activate the downstream Wnt signalling pathway. This study also presents the latest research findings on NRX and its related molecules [ABSTRACT FROM AUTHOR]

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing. [ABSTRACT FROM AUTHOR]

Neurons are highly polarized cells that possess two morphologically and functionally different types of protrusions, axons and dendrites, that function in the transmission and reception of neural signals, respectively. A great deal of attention has been paid to the specification and guidance of axons, but the mechanism of dendrite development remains mostly unknown. We report here that a polarity-regulating kinase, partitioning-defective 1 (Par1b)/microtubule affinity-regulating kinase 2 (MARK2), specifically regulates development of dendrites in hippocampal neurons. Ectopic expression of Par1b/MARK2 shortens the length and decreases branching of dendrites without significant effects on axons. Knockdown of endogenous Par1b/MARK2 by RNA interference stimulates dendrite development. Wnt stimulation and Dishevelled expression, both of which are known to induce dendrite development, induced recruitment of Par1b/MARK2 to the membrane fraction. Expression of a Par1b/MARK2 mutant, that contains a myristoylation signal and accumulates exclusively in membranes, does not affect dendrite development. In addition, Par1b/MARK2 efficiently phosphorylated MAP2, which is localized mainly in dendrites. These results indicate that Par1b/MARK2 negatively regulates dendrite development through phosphorylation of MAP2. [ABSTRACT FROM AUTHOR]

Dishevelled (Dvl) transduces signals from the Wnt receptor, Frizzled, to downstream components, leading to the stabilization of β-catenin and subsequent activation of the transcription factor T cell factor (TCF) and/or lymphoid enchancer factor (LEF). However, the mechanism of Dvl action remains unclear. Here, we report that nucleoredoxin (NRX), a thioredoxin (TRX) family protein, interacts with Dvl. Overexpression of NRX selectively suppresses the Wnt–β-catenin pathway and ablation of NRX by RNA-interference (RNAi) results in activation of TCF, accelerated cell proliferation and enhancement of oncogenicity through cooperation with mitogen-activated extracellular signal regulated kinase kinase (MEK) or Ras. We find that cells respond to H2O2 stimulation by activating TCF. Redox-dependent activation of the Wnt–β-catenin pathway occurs independently of extracellular Wnts and is impaired by RNAi of NRX . In addition, association between Dvl and NRX is inhibited by H2O2 treatment. These data suggest a relationship between the Wnt–β-catenin pathway and redox signalling through redox-sensitive association of NRX with Dvl. [ABSTRACT FROM AUTHOR]

Phosphatase of regenerating liver (PRL) is frequently overexpressed in various malignant cancers and is known to be a driver of malignancy. Here, we demonstrated that PRL overexpression causes mitotic errors that accompany spindle misorientation and aneuploidy, which are intimately associated with cancer progression. Mechanistic analyses of this phenomenon revealed dysregulation of the energy sensor kinase, AMP-activated protein kinase (AMPK), in PRL-induced mitotic errors. Specifically, immunofluorescence analysis showed that levels of phosphorylated AMPK (P-AMPK), an activated form of AMPK, at the kinetochore were reduced by PRL expression. Moreover, artificial activation of AMPK using chemical activators, such as A769662 and AICAR, in PRL-expressing cells restored P-AMPK signals at the kinetochore and normalized spindle orientation. Collectively, these results indicate the crucial importance of the activation of kinetochore-localized AMPK in the normal progression of mitosis, which is specifically perturbed by PRL overexpression.Key words: cancer, AMPK, PRL, kinetochore, mitotic errors.

Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-β pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The CNNM/CorC family proteins are Mg 2+ transporters that are widely distributed in all domains of life. In bacteria, CorC has been implicated in the survival of pathogenic microorganisms. In humans, CNNM proteins are involved in various biological events, such as body absorption/reabsorption of Mg 2+ and genetic disorders. Here, we determined the crystal structure of the Mg 2+ -bound CorC TM domain dimer. Each protomer has a single Mg 2+ binding site with a fully dehydrated Mg 2+ ion. The residues at the Mg 2+ binding site are strictly conserved in both human CNNM2 and CNNM4, and many of these residues are associated with genetic diseases. Furthermore, we determined the structures of the CorC cytoplasmic region containing its regulatory ATP-binding domain. A combination of structural and functional analyses not only revealed the potential interface between the TM and cytoplasmic domains but also showed that ATP binding is important for the Mg 2+ export activity of CorC., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Dietary magnesium (Mg 2+ ) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. Here we show that Mg 2+ feeding also enhances long-term memory in Drosophila . Normal and Mg 2+ -enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg 2+ -efflux transporter encoded by the unextended ( uex ) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex from a function in memory. Moreover, UEX localization in mushroom body Kenyon cells (KCs) is altered in memory-defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of KC Mg 2+ . We propose that regulated neuronal Mg 2+ efflux is critical for normal and Mg 2+ -enhanced memory., Competing Interests: YW, YF, EM, KJ, HM, SW No competing interests declared, (© 2020, Wu et al.)

Phosphatase of regenerating liver (PRL) is overexpressed in metastatic cancers and actively drives their malignant progression. Many studies on cultured cancer cells have implied PRL overexpression as a stimulant for cellular signaling involved in cell proliferation. However, its role in the tightly adhered and polarized epithelial cells remains largely uncharacterized. In this study, we show that inducible expression of PRL in MDCK normal epithelial cells sensitized MET, the receptor for hepatocyte growth factor (HGF), to functional activation by HGF. We found that PRL expression amplified tyrosine phosphorylation levels of various proteins, among which MET was identified to be the most abundant. This phosphorylation occurred selectively at Y1234/1235 in the activation loop of MET, whereas phosphorylation of Y1349 in the effector-binding site, which is directly involved in downstream signaling, was almost undetectable. Consistently, PRL overexpression by itself did not cause observable alterations at the cellular level. However, when cells were stimulated with HGF, phosphorylation of Y1349 was much more strongly induced in PRL-expressing cells than in control cells. This resulted in robust cell scattering and tubulogenesis, even with low levels of HGF. Collectively, these results demonstrate a unique role of PRL in regulating MET function, which is known to be crucial for remodeling of epithelial tissues and malignant progression of cancers., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Phosphatase of regenerating liver (PRL) is highly expressed in malignant cancers and promotes cancer progression. Recent studies have suggested its functional relationship with Mg 2+ , but the importance and molecular details of this relationship remain unknown. Here, we report that PRL expression is regulated by Mg 2+ and PRL protects cells from apoptosis under Mg 2+ -depleted conditions. When cultured cells were subjected to Mg 2+ depletion, endogenous PRL protein levels increased significantly. siRNA-mediated knockdown of endogenous PRL did not significantly affect cell proliferation under normal culture conditions, but it increased cell death after Mg 2+ depletion. Imaging analyses with a fluorescent probe for Mg 2+ showed that PRL knockdown severely reduced intracellular Mg 2+ levels, indicating a role for PRL in maintaining intracellular Mg 2+ We also examined the mechanism of augmented expression of PRL proteins and found that PRL mRNA transcription was stimulated by Mg 2+ depletion. A series of analyses revealed the activation and the crucial importance of signal transducer and activator of transcription 1 in this process. Collectively, these results implicate PRL in maintaining cellular Mg 2+ homeostasis., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)