Recovery from the damage of cranial radiation modulated by memantine, an NMDA receptor antagonist, combined with hyperbaric oxygen therapy.

BACKGROUND: Radiotherapy is an important treatment option for central nervous system malignancies. However, cranial radiation induces hippocampal dysfunction and white matter injury; this leads to cognitive dysfunction, and results in a reduced quality of life in patients. Excitatory glutamate signaling through N-methyl-d-aspartate receptors (NMDARs) plays a central role both in hippocampal neurogenesis and in the myelination of oligodendrocytes in the cerebrum. METHODS: We provide a method for quantifying neurogenesis in human subjects in live brain during cancer therapy. Neuroimaging using originally created behavioral tasks was employed to examine human hippocampal memory pathway in patients with brain disorders. RESULTS: Treatment with memantine, a non-competitive NMDAR antagonist, reversed impairment in hippocampal pattern separation networks as detected by functional magnetic resonance imaging. Hyperbaric preconditioning of the patients just before radiotherapy with memantine mostly reversed white matter injury as detected by whole brain analysis with Tract-Based Spatial Statics. Neuromodulation combined with the administration of hyperbaric oxygen therapy and memantine during radiotherapy facilitated the restoration of hippocampal function and white matter integrity, and improved higher cognitive function in patients receiving cranial radiation. CONCLUSIONS: The method described herein, for diagnosis of hippocampal dysfunction, and therapeutic intervention can be utilized to restore some of the cognitive decline experienced by patients who have received cranial radiation. The underlying mechanism of restoration is the production of new neurons, which enhances functionality in pattern separation networks in the hippocampi, resulting in an increase in cognitive score, and restoration of microstructural integrity of white matter tracts revealed by Tract-Based Spatial Statics Analysis.

Generation of four iPSC lines from a family harboring a 1p36-35 haplotype linked with bipolar disorder and recurrent depressive disorder: Three-generation patients and a healthy sibling.

Induced pluripotent stem cells (iPSCs) obtained from genetically characterized patients benefit the biological study of bipolar disorder (BD). Here, we present iPSC lines from three-generation patients with BD and recurrent depressive disorder (RDD) and a healthy control sibling in a family. All patients shared the specified haplotype in the 1p36-35, previously reported as the susceptibility locus of mood disorders. iPSCs were generated with the reprogramming factors OTC3/4, l-MYC, LIN28, SOX2, KLF4, and p53 shRNA through non-integrated episomal vectors. All iPSC lines strongly expressed pluripotency markers and proved the ability to differentiate into three germ lineages in vitro.

Ancient genomes from the last three millennia support multiple human dispersals into Wallacea.

Previous research indicates that human genetic diversity in Wallacea-islands in present-day Eastern Indonesia and Timor-Leste that were never part of the Sunda or Sahul continental shelves-has been shaped by complex interactions between migrating Austronesian farmers and indigenous hunter-gatherer communities. Yet, inferences based on present-day groups proved insufficient to disentangle this region's demographic movements and admixture timings. Here, we investigate the spatio-temporal patterns of variation in Wallacea based on genome-wide data from 16 ancient individuals (2600-250 years BP) from the North Moluccas, Sulawesi and East Nusa Tenggara. While ancestry in the northern islands primarily reflects contact between Austronesian- and Papuan-related groups, ancestry in the southern islands reveals additional contributions from Mainland Southeast Asia that seem to predate the arrival of Austronesians. Admixture time estimates further support multiple and/or continuous admixture involving Papuan- and Asian-related groups throughout Wallacea. Our results clarify previously debated times of admixture and suggest that the Neolithic dispersals into Island Southeast Asia are associated with the spread of multiple genetic ancestries.

Altered Calcium Permeability of AMPA Receptor Drives NMDA Receptor Inhibition in the Hippocampus of Murine Obesity Models.

Evidence has accumulated that higher consumption of high-fat diets (HFDs) during the juvenile/adolescent period induces altered hippocampal function and morphology; however, the mechanism behind this phenomenon remains elusive. Using high-resolution structural imaging combined with molecular and functional interrogation, a murine model of obesity treated with HFDs for 12 weeks after weaning mice was shown to change in the glutamate-mediated intracellular calcium signaling and activity, including further selective reduction of gray matter volume in the hippocampus associated with memory recall disturbance. Dysregulation of intracellular calcium concentrations was restored by a non-competitive α-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) antagonist, followed by normalization of hippocampal volume and memory recall ability, indicating that AMPARs may serve as an attractive therapeutic target for obesity-associated cognitive decline.

Haplotype phasing of a bipolar disorder pedigree revealed rare multiple mutations of SPOCD1 gene in the 1p36-35 susceptibility locus.

BACKGROUND: The etiology of bipolar disorder (BD) is poorly understood. Considering the complexity of BD, pedigree-based sequencing studies focusing on haplotypes at specific loci may be practical to discover high-impact risk variants. This study comprehensively examined the haplotype sequence at 1p36-35 BD and recurrent depressive disorder (RDD) susceptibility loci. METHODS: We surveyed BD families in Okinawa, Japan. We performed linkage analysis and determined the phased sequence of the affected haplotype using whole genome sequencing. We filtered rare missense variants on the haplotype. For validation, we conducted a case-control genetic association study on approximately 3000 Japanese subjects. RESULTS: We identified a three-generation multiplex pedigree with BD and RDD. Strikingly, we identified a significant linkage with mood disorders (logarithm of odds [LOD] = 3.61) at 1p36-35, supported in other ancestry studies. Finally, we determined the entire sequence of the 6.4-Mb haplotype shared by all affected subjects. Moreover, we found a rare triplet of missense variants in the SPOCD1 gene on the haplotype. Notably, despite the rare frequency, one heterozygote with multiple SPOCD1 variants was identified in an independent set of 88 BD type I genotyping samples. LIMITATIONS: The 1p36-35 sequence was obtained from only a single pedigree. The replicate sample was small. Short-read sequencing might miss structural variants. A polygenic risk score was not analyzed. CONCLUSION: The 1p36-35 haplotype sequence may be valuable for future BD variant studies. In particular, SPOCD1 is a promising candidate gene and should be validated.

The crucial role of the TRPM7 kinase domain in the early stage of amelogenesis.

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

Tescalcin is a potential target of class I histone deacetylase inhibitors in neurons.

Class I histone deacetylase (HDAC) inhibitors are believed to have positive effects on neurite outgrowth, synaptic plasticity, and neurogenesis in adult brain. However, the downstream molecular targets of class I HDAC inhibitors in neurons are not clear. Although class I HDAC inhibitors are thought to broadly promote transcription of many neuronal genes through enhancement of histone acetylation, the affected gene set may include unidentified genes that are essential for neuronal survival and function. To identify novel genes that are targets of class I HDAC inhibitors, we used a microarray to screen transcripts from neuronal cultures and evaluated changes in protein and mRNA expression following treatment with four HDAC inhibitors. We identified tescalcin (Tesc) as the most strongly up-regulated gene following treatment with class I HDAC inhibitors in neurons. Moreover, hippocampal neurons overexpressing TESC showed a greater than 5-fold increase in the total length of neurites and number of branch points compared with controls. These findings highlight a potentially important role for TESC in mediating the neuroprotective effect of class I HDAC inhibitors. TESC may also be involved in the development of brain and neurodegenerative diseases through epigenetic mechanisms.

Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone.

There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body's immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography-tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies.

Advanced maritime adaptation in the western Pacific coastal region extends back to 35,000-30,000 years before present.

Maritime adaptation was one of the essential factors that enabled modern humans to disperse all over the world. However, geographic distribution of early maritime technology during the Late Pleistocene remains unclear. At this time, the Indonesian Archipelago and eastern New Guinea stand as the sole, well-recognized area for secure Pleistocene evidence of repeated ocean crossings and advanced fishing technology. The incomplete archeological records also make it difficult to know whether modern humans could sustain their life on a resource-poor, small oceanic island for extended periods with Paleolithic technology. We here report evidence from a limestone cave site on Okinawa Island, Japan, of successive occupation that extends back to 35,000-30,000 y ago. Well-stratified strata at the Sakitari Cave site yielded a rich assemblage of seashell artifacts, including formally shaped tools, beads, and the world's oldest fishhooks. These are accompanied by seasonally exploited food residue. The persistent occupation on this relatively small, geographically isolated island, as well as the appearance of Paleolithic sites on nearby islands by 30,000 y ago, suggest wider distribution of successful maritime adaptations than previously recognized, spanning the lower to midlatitude areas in the western Pacific coastal region.

Identification of a novel cell-penetrating peptide targeting human glioblastoma cell lines as a cancer-homing transporter.

Cell-penetrating peptides (CPPs) as a novel biomedical delivery system have been highly anticipated, since they can translocate across biological membranes and are capable of transporting their cargo inside live cells with minimal invasiveness. However, non-selective internalization in various cell types remains a challenge in the clinical application of CPPs, especially in cancer treatment. In this study, we attempted to identify novel cancer-homing CPPs to target glioblastoma multiforme (GBM), which is often refractory and resistant to treatment. We screened for CPPs showing affinity for the human GBM cell line, U87MG, from an mRNA display random peptide library. One of the candidate peptides which amino-acid sequence was obtained from the screening showed selective cell-penetrating activity in U87MG cells. Conjugation of the p16(INK4a) functional peptide to the GBM-selective CPP induced cellular apoptosis and reduced phosphorylated retinoblastoma protein levels. This indicates that the CPP was capable of delivering a therapeutic molecule into U87MG cells inducing apoptosis. These results suggest that the novel CPP identified in this study permeates with high affinity into GBM cells, revealing it to be a promising imaging and therapeutic tool in the treatment of glioblastoma.

Inactivation of TRPM7 kinase activity does not impair its channel function in mice.

Transient receptor potential (TRP) family channels are involved in sensory pathways and respond to various environmental stimuli. Among the members of this family, TRPM7 is a unique fusion of an ion channel and a C-terminus kinase domain that is highly expressed in immune cells. TRPM7 serves as a key molecule governing cellular Mg(2+) homeostasis in mammals since its channel pore is permeable to Mg(2+) ions and can act as a Mg(2+) influx pathway. However, mechanistic links between its kinase activity and channel function have remained uncertain. In this study, we generated kinase inactive knock-in mutant mice by mutagenesis of a key lysine residue involved in Mg(2+)-ATP binding. These mutant mice were normal in development and general locomotor activity. In peritoneal macrophages isolated from adult animals the basal activity of TRPM7 channels prior to cytoplasmic Mg(2+) depletion was significantly potentiated, while maximal current densities measured after Mg(2+) depletion were unchanged in the absence of detectable kinase function. Serum total Ca(2+) and Mg(2+) levels were not significantly altered in kinase-inactive mutant mice. Our findings suggest that abolishing TRPM7 kinase activity does not impair its channel activity and kinase activity is not essential for regulation of mammalian Mg(2+) homeostasis.

Novel function of MKP-5/DUSP10, a phosphatase of stress-activated kinases, on ERK-dependent gene expression, and upregulation of its gene expression in colon carcinomas.

Mitogen-activated protein kinase phosphatase 5 (MKP-5)/DUSP10 acts as a phosphatase of stress-activated kinases (JNK and p38), but its activity towards ERK has not been demonstrated. In the present study we observed that MKP-5 interacts with ERK, retains it in the cytoplasm, suppresses its activation and downregulates ERK-dependent transcription. These data suggested a novel MKP-5 function as a scaffold protein for the ERK pathway. We analyzed MKP-5 gene expression in several tumors, and found that it is frequently upregulated in colorectal but not in lung and breast cancer, suggesting its association with the malignant phenotype of colon cancer.

DUSP13B/TMDP inhibits stress-activated MAPKs and suppresses AP-1-dependent gene expression.

The dual-specificity phosphatase (DUSP) 13 gene encodes two atypical DUSPs, DUSP13B/TMDP and DUSP13A/MDSP using alternative exons. DUSP13B protein is most highly expressed in testis, particularly in spermatocytes and round spermatids of the seminiferous tubules, while that of DUSP13A is restricted to skeletal muscle. Here, we show that DUSP13B inactivated MAPK activation in the order of selectivity, JNK = p38>ERK in cells, while DUSP13A did not show MAPK phosphatase activity. Reporter gene analysis showed that DUSP13B had significant inhibitory effect on AP-1-dependent gene expression, but DUSP13A did not. To our knowledge, DUSP13B is the first identified testis-specific phosphatase that inhibits stress-activated MAPKs. These data suggest an important role for DUSP13B in protection from external stress during spermatogenesis.

MKP-7, a JNK phosphatase, blocks ERK-dependent gene activation by anchoring phosphorylated ERK in the cytoplasm.

MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localized with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.

Phosphorylation of Ser-446 determines stability of MKP-7.

MAPK cascades can be negatively regulated by members of the MAPK phosphatase (MKP) family. However, how MKP activity is regulated is not well characterized. MKP-7, a JNK-specific phosphatase, possesses a unique COOH-terminal stretch (CTS) in addition to domains conserved among MKP family members. The CTS contains several motifs such as a nuclear localization signal, a nuclear export signal, PEST sequences, and a serine residue (Ser-446) that can be phosphorylated by activated ERK, suggesting an important regulatory role(s).(35)S-pulse labeling experiments indicate that the half-life of MKP-7 is 1.5 h, a period significantly elongated by deleting the CTS. We also show that overexpressed MKP-7 is polyubiquitinated when co-expressed with ubiquitin and that proteasome inhibitors markedly inhibit MKP-7 degradation. We also determined that MKP-7 phosphorylated at Ser-446 has a longer half-life than unphosphorylated form of the wild type protein, as does a phospho-mimic mutant of MKP-7. These results indicate that activation of the ERK pathway strongly blocks JNK activation through stabilization of MKP-7 mediated by phosphorylation.

Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446.

We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK >> p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK.

Oviductin, the oviductal protease that mediates gamete interaction by affecting the vitelline coat in Bufo japonicus: its molecular cloning and analyses of expression and posttranslational activation.

Previous studies indicated that the acquisition of egg fertilizability during transit through the pars recta portion of the oviduct in Bufo japonicus is accompanied by hydrolytic conversion of the vitelline coat 40- to 52-kDa components to 39-kDa components induced by a 66-kDa serine protease, "oviductin." In this study, we cloned a 3028-bp cDNA that contained an open reading frame encoding 974 amino acids with a calculated molecular mass of 107.6 kDa, including two protease domains and three repeats of CUB domains. Sequence analysis indicated that the catalytically active 66-kDa protein comprised an N-terminally located oviductin protease and two CUB domains. The oviductin gene was transcribed as a part of 6-kb mRNA that was expressed specifically in the cells lining the bottom of epithelial folds in the oviductal pars recta, and this expression was highly accelerated when the pars recta fragments were cultured in the presence of hCG. Western blot analyses using antibodies against a protease domain revealed that the catalytically inactive 102-kDa proteins in the pars recta granules yield 66-kDa catalytically active and 82- and 59-kDa inactive molecules. We propose that the oviductin translated as 107.6-kDa precursors are processed both N- and C-terminally to give rise to a 66-kDa active form comprising a serine protease and two CUB domains.

Heart formative factor(s) is localized in the anterior endoderm of early Xenopus neurula.

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)+ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).

Relative amounts of basic nuclear proteins SP4 and SP5 in Xenopus laevis sperm correlate with gene copy number.

Mature sperm of an anuran amphibian Xenopus laevis contain six sperm-specific basic nuclear proteins (SP 1-6). In an attempt to understand how the amounts of SP produced are regulated during transcription and translation, the relative amounts of proteins and messenger RNA (mRNA), and the gene number in nuclei were compared between SP4 and SP5. Measurements of the peak areas of proteins separated by reversed-phase high performance liquid chromatography (HPLC) indicated that SP4 is present in about a five times greater amount than SP5. Nuclease S1 protection assays showed the existence of SP4 mRNA in about a five times greater amount than SP5 mRNA. Southern hybridization analyses of restriction enzyme-digested genomic DNA indicated a single copy of SP5 gene in the haploid genome, as contrasted with the five SP4 genes found in our previous study. Thus the deposition of different amounts of SP4 and SP5 in sperm nuclei reflects the relative number of these genes in the genome.

Involvement of carbohydrate moieties of the toad egg vitelline coat in binding with fertilizing sperm.

Vitelline coats (VC) were isolated from the eggs of Bufo japonicus, and were added with sperm in reconstituted salt solution, which mimics the physiological role of jelly envelopes, to determine the rates of sperm binding per unit area (0.2 mm2 ) of VC. The rate of sperm binding to VC from uterine eggs was high, but was low to VC from coelomic eggs and eggs activated in 1/20 De Boer's solution (DB) and moderately low to VC from eggs activated in DB. The binding rate increased when VC from coelomic eggs were treated with extracts of the pars recta portion of the oviduct. The sperm that bound to VC were not acrosome-reacted and their binding to VC required both a low salinity, assuring motility of sperm, and sufficiently high levels of Ca2+ and Mg2+ . The rate of sperm binding was reduced by either coexisting solubilized VC materials, periodate-oxidation of VC or the pretreatment of VC with Fab fragments of anti-VC antibodies, which reacted mostly to carbohydrate residues of VC glycoproteins. Sperm-VC binding assays in combination with gel-filtrated VC components revealed that the fractions containing 36-39 kDa components were most effective both in inhibiting the binding and in neutralizing the antibody induced inhibition of binding. These results indicate that carbohydrate moieties in 36-39 kDa glycoproteins of VC, exposed as a result of hydrolysis by the oviducal pars recta protease, are involved in binding with fertilizing sperm.