Loss of Ftsj1 perturbs codon-specific translation efficiency in the brain and is associated with X-linked intellectual disability.

FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1) gene has been implicated in X-linked intellectual disability (XLID), but the molecular pathogenesis is unknown. We show that Ftsj1 is responsible for 2'-O-methylation of 11 species of cytosolic transfer RNAs (tRNAs) at the anticodon region, and these modifications are abolished in Ftsj1 knockout (KO) mice and XLID patient-derived cells. Loss of 2'-O-methylation in Ftsj1 KO mouse selectively reduced the steady-state level of tRNAPhe in the brain, resulting in a slow decoding at Phe codons. Ribosome profiling showed that translation efficiency is significantly reduced in a subset of genes that need to be efficiently translated to support synaptic organization and functions. Ftsj1 KO mice display immature synaptic morphology and aberrant synaptic plasticity, which are associated with anxiety-like and memory deficits. The data illuminate a fundamental role of tRNA modification in the brain through regulation of translation efficiency and provide mechanistic insights into FTSJ1-related XLID.

Prognostic impact of residual lateral lymph node metastasis after neoadjuvant (chemo)radiotherapy in patients with advanced low rectal cancer.

Background: There is a lack of large studies focusing on the prognostic significance of lateral lymph node (LLN) metastasis following LLN dissection (LLND) in rectal cancer. The aim of this study was to evaluate the prognostic impact of LLN metastases on survival of patients with advanced low rectal cancer. Methods: Consecutive patients with locally advanced, but not metastatic, extraperitoneal rectal cancer treated with neoadjuvant (chemo)radiotherapy plus total mesorectal excision between 2004 and 2015 were included in the study. LLND was performed when pretreatment imaging documented enlarged LLNs (7 mm or greater in size). Localization of nodal metastases and long-term outcomes were analysed. Kaplan-Meier analysis was used to compare the survival of patients with ypN0 disease with that of patients with mesorectal ypN+/LLN- status and patients with positive LLNs. The Cox proportional hazards model was used to evaluate predictors of disease-free survival (DFS) and local recurrence. Results: A total of 613 patients were included in the study; LLND was performed in 212 patients (34·6 per cent) and 57 (9·3 per cent) had LLN metastasis. Patients with LLN metastasis had improved DFS and local recurrence cumulative incidence rates compared with patients with mesorectal ypN2+/LLN- disease (DFS: P = 0·014; local recurrence: P = 0·006). Although the DFS rate of patients with LLN metastasis was worse than that of patients with ypN0 disease (P < 0·001), the cumulative incidence of local recurrence was similar (P = 0·491). In multivariable analysis, residual LLN metastasis was not an independent predictor of worse DFS or local recurrence. Conclusion: LLN metastasis is not an independent predictor of local recurrence or survival. Survival of patients presenting with LLN metastasis after (chemo)radiotherapy was intermediate between that of patients with ypN0 status and those with mesorectal ypN2 positivity.

Antecedentes: No existen en la literatura grandes estudios dirigidos a investigar la importancia pronóstica de las metástasis en los ganglios linfáticos laterales (lateral lymph nodes, LLN) después de la disección de los mismos (LLN dissection, LLND) en pacientes con cáncer de recto. El objetivo de este estudio fue evaluar el impacto pronóstico de las metástasis en los LLN sobre la supervivencia de los pacientes con cáncer de recto. Métodos: Se analizaron 613 pacientes consecutivos con cáncer de recto localmente avanzado extraperitoneal y no metastásico tratados con (quimio)radioterapia neoadyuvante seguida de resección total del mesorrecto (total mesorectal excision, TME) entre 2004 y 2015. Se realizó una LLND cuando el estudio mediante pruebas de imagen previo el tratamiento mostró LLN aumentados de tamaño ≥ 7 mm. Se analizó la localización de las metástasis ganglionares y los resultados a largo plazo. El análisis de supervivencia se realizó mediante el método de Kaplan­Meier para comparar las supervivencias de los pacientes ypN0 frente a los pacientes ypN con positividad mesorrectal/LLN negativos y frente a los pacientes LLN positivos. Se utilizó el modelo de riesgo proporcional de Cox para evaluar los factores predictivos de supervivencia libre de enfermedad y de recidiva local. Resultados: Se realizó una LLND en 212 (34,6%) pacientes, y 57 (9,3%) pacientes presentaban metástasis en los LLN. Los pacientes con metástasis en los LLN presentaron mejores curvas de incidencia acumulada de recidiva local y de supervivencia libre de enfermedad en comparación con los pacientes con ganglios mesorrectales ypN2 positivos/LLN negativos (respectivamente, P = 0,0135 y P = 0,0060). Aunque la curva de la supervivencia libre de enfermedad de los pacientes con metástasis en los LLN fue peor que la de los pacientes ypN0 (P < 0,0001), la incidencia acumulada de recidiva local fue similar (P = 0,4905). En el análisis multivariable, la metástasis residual en los LLN no fue un factor predictivo independiente de peor supervivencia libre de enfermedad ni de recidiva local. Conclusión: Las metástasis en los LLN no es un factor predictivo independiente de recidiva local o supervivencia. Los pacientes que presentaron metástasis en los LLN después de (quimio)radioterapia mostraron características de supervivencia intermedias entre ypN0 y pacientes con ganglios mesorrectales ypN2 positivos.

Protein phosphatase 1 modulation of neostriatal AMPA channels: regulation by DARPP-32 and spinophilin.

Modulation of AMPA-type glutamate channels is important for synaptic plasticity. Here we provide physiological evidence that the activity of AMPA channels is regulated by protein phosphatase 1 (PP-1) in neostriatal neurons and identify two distinct molecular mechanisms of this regulation. One mechanism involves control of PP-1 catalytic activity by DARPP-32, a dopamine- and cAMP-regulated phosphoprotein highly enriched in neostriatum. The other involves binding of PP-1 to spinophilin, a protein that colocalizes PP-1 with AMPA receptors in postsynaptic densities. The results suggest that regulation of anchored PP-1 is important for AMPA-receptor-mediated synaptic transmission and plasticity.

A high-efficiency protein transduction system demonstrating the role of PKA in long-lasting long-term potentiation.

Proteins and peptides have been demonstrated to penetrate across the plasma membrane of eukaryotic cells by protein transduction domains. We show that protein transduction by 11 arginine (11R) is an efficient method of delivering proteins into the neurons of brain slices. Here, we demonstrate that PKA inhibitory peptide, fused with 11R and nuclear localization signal, delivers the peptide exclusively into the nuclear compartment of neurons in brain slices. This inhibitory peptide blocked both cAMP responsive element-binding protein phosphorylation and long-lasting long-term potentiation (LTP) induction, but not early LTP. These results highlight transduction of proteins and peptides into specific neuronal subcellular compartments in brain slices as a powerful tool for studying neuronal plasticity.

Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8.

The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined. The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid. Most of the peptides were sequenced by solid phase Edman degradation. Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion. The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.

Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau.

Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.

A novel tau-tubulin kinase from bovine brain.

During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, beta-tubulin, MAP2 and alpha-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.

Thioredoxin peroxidase in the Cyanobacterium Synechocystis sp. PCC 6803.

The amino acid sequence deduced from the open reading frame designated sll0755 in Synechocystis sp. PCC 6803 is similar to the amino acid sequences of thioredoxin peroxidases from other organisms. In the present study, we found that a recombinant SLL0755 protein that was expressed in Escherichia coli was able to reduce H2O2 and tertiary butyl hydroperoxide with thioredoxin from E. coli as the electron donor. Targeted disruption of open reading frame sll0755 in Synechocystis sp. PCC 6803 cells completely eliminated the H2O2-dependent and tertiary butyl hydroperoxide-dependent photosynthetic evolution of oxygen and the electron flow in photosystem II. These results indicate that the product of open reading frame sll0755 is a thioredoxin peroxidase whose activities are coupled to the photosynthetic electron transport system in Synechocystis sp. PCC 6803.

Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts.

To analyze the potential of the active oxygen-scavenging system of chloroplasts, we introduced Escherichia coli catalase into tobacco chloroplasts. Photosynthesis of transgenic plants was tolerant to high irradiance under drought conditions, while the wild plants suffered severe damage in photosynthesis under the same conditions. Irrespective of responses to the stress, ascorbate peroxidase was completely inactivated both in the transgenic and wild-type plants. These findings are contrary to the established idea that the ascorbate peroxidase-mediated antioxidative system protects chloroplasts from oxidative stress.

A novel brain-specific 25 kDa protein (p25) is phosphorylated by a Ser/Thr-Pro kinase (TPK II) from tau protein kinase fractions.

A novel brain-specific 25 kDa protein (p25) was purified from a bovine brain extract. The protein was phosphorylated by Ser/Thr-Pro kinase (TPK II) in tau protein kinase fractions at the Ser residues of Ser-Pro sequences. Using immunoblot analysis, the protein was found only in brain extracts, and was most abundant in the brain regions such as cerebrum and hippocampus, but less abundant in cerebellum, medulla oblongata and olfactory bulb. The protein was detected in rat, bovine and human brain extracts, indicating that this protein specifically exists in mammalian brain tissues.

Molecular cloning of cDNAs encoding two gonadotrophin beta subunits (GTH-I beta and -II beta) from the masu salmon, Oncorhynchus masou: rapid divergence of the GTH-I beta gene.

Two types of cDNA (GTH-I beta and -II beta) encoding the beta subunit of masu salmon (Oncorhynchus masou) gonadotrophin were cloned using the reverse transcription-polymerase chain reaction for pituitary mRNAs. The nucleotide sequence of GTH-I beta cDNA was 469 bp long, encoding 137 amino acids, and GTH-II beta cDNA was 476 bp long, encoding 142 amino acids. These two masu salmon beta subunit types showed low homologies of 52% (nucleotide sequence) and 33% (amino acid sequence). The evolutionary interval between masu and chum salmon was estimated to be 5.65 and 1.43 million years by comparing GTH-I beta and GTH-II beta respectively. These time values are markedly inconsistent with the evolutionary time (3.0 million years) estimated from fossil records and an isozyme study. Southern blot analyses showed that the I beta gene restriction fragment lengths differed among five teleosts, whereas, with one exception, the II beta gene showed well conserved patterns. Therefore, the GTH-I beta gene may have diverged at a faster rate than the GTH-II beta gene.

Molecular cloning of cDNAs encoding two types of gonadotrophin alpha subunit from the masu salmon, Oncorhynchus masou: construction of specific oligonucleotides for the alpha 1 and alpha 2 subunits.

Two types of cDNA (GTH alpha 1 and -alpha 2) encoding the alpha subunits of masu salmon (Oncorhynchus masou) gonadotrophin were cloned by the reverse transcription-polymerase chain reaction for pituitary mRNAs. The nucleotide sequences showed that the GTH alpha 1 cDNA was 380 bp long, encoding 119 amino acids, and that GTH alpha 2 cDNA was 365 bp long, encoding 114 amino acids. The masu salmon alpha subunit types had a few differences between the sequences, with homologies of 80% (nucleotide sequence) and 72% (amino acid sequence). The structural difference between the alpha 1 and alpha 2 subunits was predicted using hydropathic analysis. The evolutionary interval between masu and chum salmon was estimated to be 4.0 and 2.3 million years by comparing their GTH alpha 1 and -alpha 2 subunits respectively. These time values are roughly consistent with the evolutionary time interval (3.0 million years) estimated from fossil records and an isozyme study. Specific synthetic oligonucleotide probes were constructed and used for genomic Southern blot analyses. The restriction fragment sizes of the GTH alpha 1 and -alpha 2 genes were similar, and when their patterns were compared with those from four other teleosts, each species showed a different pattern from the others, but no difference between their respective alpha 1 and alpha 2 genes. Therefore, the structural features of the GTH alpha 1 and -alpha 2 genes may have diverged in a similar manner in these five teleosts.

Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector.

Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein alpha and FSH beta subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.

An immunosuppressant, FK506, protects against neuronal dysfunction and death but has no effect on electrographic and behavioral activities induced by systemic kainate.

Kainate is a potent agonist of an excitatory amino acid receptor subtype in the central nervous system, and causes neuronal death in several regions of the brain. Neurons are preferentially killed in the hippocampus, especially in the CA1 region, by systemic administration of kainate. It is speculated that functional alterations occur in the neurons preceding death. We examined the effect of FK506 on kainate-induced neuronal death and functional alterations in the rat hippocampal CA1 region. FK506 had no effect on electrographic and behavioral seizure activities induced by kainate; however, it prevented neuronal death measured seven days after administration. Although neither death nor morphological alterations of neurons were observed in the CA1 region 24 h after administration, the neurons exhibited decreased excitatory postsynaptic potentials and enhanced long-term potentiation. This functional alteration was not detected in the rats administered FK506 prior to kainate. Taken together, these observations indicate that functional alteration precedes neuronal death in rats systemically administered kainate and that FK506 prevents both. It is suggested that FK506 exerts its neuroprotective effect not by attenuating electrographic and behavioral seizure activities, but by protecting neurons from kainate-induced functional disorders.

Localization and developmental changes in the neuron-specific cyclin-dependent kinase 5 activator (p35nck5a) in the rat brain.

Mammalian brains contain a cde2-like protein kinase which is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a brain-specific regulatory subunit with a molecular weight of 35,000. In this study, we examined the temporal and spatial expression patterns of p35nck5a in the developing rat brain. Northern blot analysis showed that p35nck5a messenger RNA expression was low in the brain of 12-day postcoitum rats, and increased to a much higher level from 18 days postcoitum to two weeks after birth, and then declined at three weeks after birth. These developmental changes in p35nck5a expression correlated with the changes in Cdk5-associated kinase activity during brain development. These data suggest that p35nck5a is the specific activator for Cdk5 in the brain. Immunohistochemical and in situ hybridization studies demonstrated the presence of p35nck5a protein in postmitotic neurons but not in glial cells at all stages of brain development, indicating that p35nck5a is a neuron-specific protein. In the adult brain, the protein was rich in cell bodies and dendrites, and only very low amounts were detected in axons. In fetal and neonatal brains, however, axonal pathways such as the corpus callosum and external capsule were also stained with anti-p35nck5a antibody. Our findings suggest that p35nck5a is neuron specific, and a specific activator for Cdk5, and the subcellular localization of the two is strictly regulated depending on brain development. Neuronal Cdc2-like kinase may play key roles in neuronal maturation, synaptic formation, and neuronal plasticity.

Multiple binding sites for nuclear proteins of the anterior pituitary are located in the 5'-flanking region of the porcine follicle-stimulating hormone (FSH) beta-subunit gene.

Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), are synthesized specifically in the gonadotropes of the anterior pituitary. The aim of this study was to investigate nuclear factors that bind specifically to the porcine FSH beta-subunit gene. We examined nuclear protein binding to 2.75 kilobase pairs (kbp) of DNA adjacent to the porcine FSH beta-subunit gene: about 2.32 kbp of upstream DNA and 0.43 kbp of downstream DNA. The upstream region contains only TATA box, CACCC element, and some imperfect sequences of cAMP-responsive element, activator protein-1 binding site, and activator protein-2 binding site. Gel mobility shift assay using nuclear proteins extracted from the porcine anterior pituitary revealed that the proteins bound to a limited region of DNA, 107 bp long (designated as Fd2), located about -800 bp upstream from the transcription initiation site. Competitive binding assays demonstrated that the protein binding was sequence specific; the addition of excess amounts of several putative regulatory sequences and plasmid (non-homologous) DNA fragments did not reduce the binding. Furthermore, all five subfragments of Fd2 were also bound by the pituitary nuclear proteins, showing that the entire region of Fd2 is involved in this interaction. Southwestern blotting demonstrated that at least seven protein species of 110, 98, 78, 63, 52, 42, and 35 kDa recognize Fd2. Nuclear proteins from several other porcine tissues were also able to bind to the Fd2 fragment but the gel shift patterns were different and the bindings were weak, although only the cerebellum showed a pattern of binding that was similar to that of the anterior pituitary. These data suggest that multiple proteins of the anterior pituitary recognize a specific region of the porcine FSH beta-subunit gene.

Presence of activating transcription factor 4 (ATF4) in the porcine anterior pituitary.

The two-hybrid system that identifies protein-protein interactions in a yeast expression system was used to investigate porcine anterior pituitary transcription factors. Four cDNA clones of a protein interacting with the leucine zipper domain of porcine cJun were obtained. Their nucleotide sequences revealed that they encode activating transcription factor 4 (ATF4). A full-length cDNA of porcine ATF4 was obtained by the polymerase chain reaction, and its deduced amino acid sequence showed 88 and 83% identity to human and mouse ATF4, respectively. Reverse transcription-polymerase chain reaction analysis of mRNAs prepared from 11 porcine tissues demonstrated that ATF4 is ubiquitous. Immunohistochemistry showed that ATF4 is present in the hormone producing cells of the anterior pituitary, but absent in some cells of the anterior pituitary. Further binding analysis revealed that ATF4 also interacts with itself and cFos. This evidence of ATF4 homodimerization, as well as heterodimerization with cJun and cFos in the anterior pituitary suggests a novel mechanism for the regulation of gene expression in this tissue.

Developmental alteration and neuron-specific expression of bone morphogenetic protein-6 (BMP-6) mRNA in rodent brain.

Bone morphogenetic proteins (BMPs) are a group of proteins which induce bone formation from mesenchymal cells. The existence of BMPs in the nervous system as well as in bone tissue has recently been reported. In this study, we show that BMP-6 is neuron-specific, and describe the temporal and spatial expression patterns of BMP-6 mRNA in the developing rat and gerbil brain. Northern blot analysis showed that the BMP-6 transcript level was specifically high from newborn to 3 weeks after birth compared with those in fetal and adult rats. In situ hybridization showed that most of the neurons possessed high levels of BMP-6 mRNA in the neonatal brain, while in the adult brain, BMP-6 mRNA level was significantly decreased in most of the neurons except those in hippocampus which retained high levels. Furthermore, to show that the BMP-6 expression was specific to neurons, we induced delayed neuronal cell death and compensative glial cell proliferation in the gerbil hippocampus by transient ischemia. Our findings collectively suggest that BMP-6 is neuron-specific and may play important roles in neuronal maturation and synapse formation.