In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

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.

Structural features of Ca2+/calmodulin-dependent protein kinase II revealed by electron microscopy.

The molecular conformation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the rat forebrain and cerebellum was studied by means of EM using a quick-freezing technique. Each molecule appeared to be composed of two kinds of particles, with one larger central particle and smaller peripheral particles and had shapes resembling that of a flower with 8 or 10 "petals". A favorable shadowing revealed that each peripheral particle had a thin link to the central particle. We predicted that the 8-petal molecules and 10-petal molecules were octamers and decamers of CaM kinase II subunits, respectively, each assembled with the association domains of subunits gathered in the center, and the catalytic domains in the peripheral particles. Binding of antibodies to the enzyme molecules suggested that molecules with 8 and 10 peripheral particles were homopolymers composed only of beta subunit and of alpha subunit, respectively, specifying that CaM kinase II consists of homopolymer of either alpha or beta subunits.

Structural features of membrane fusion between influenza virus and liposome as revealed by quick-freezing electron microscopy.

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23 degrees C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10-20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or "microprotrusions of the lipid bilayer." At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the "initial fusion pore" at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.

Minerals and Trace Elements in Milk, Milk Products, Infant Formula, and Adult/Pediatric Nutritional Formula, ICP-MS Method: Collaborative Study, AOAC Final Action 2015.06, ISO/DIS 21424, IDF 243.

AOAC Final Action Official MethodSM 2015.06 "Minerals and Trace Elements in Milk, Milk Products, Infant Formula and Adult/Pediatric Nutritional Formula, ICP-MS Method" was collaboratively studied. Note that "milk, milk products" has now been added to the title of the Final Action method because whole milk and several dairy ingredients were successfully incorporated into the collaborative study for the purpose of developing an International Organization for Standardization/International Dairy Federation standard (ISO/DIS 21424; in progress). The method determines sodium, magnesium, phosphorus, potassium, calcium, iron, manganese, zinc, copper, chromium, molybdenum, and selenium by inductively coupled plasma (ICP)-MS after microwave digestion. Ten laboratories participated in the study, and data from five different model ICP-MS units were represented. Thirteen products, five placebo products, and six dairy samples were tested as blind duplicates in this study, along with a standard reference material, for a total 50 samples. The overall repeatability and reproducibility for all samples met Standard Method Performance Requirements put forth by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals, with a few exceptions. Comparisons are made to ICP-atomic emission data from a collaborative study of AOAC Official Method 2011.14 carried out concurrently on these same samples.

Certified reference materials for radionuclides in Bikini Atoll sediment (IAEA-410) and Pacific Ocean sediment (IAEA-412).

The preparation and characterization of certified reference materials (CRMs) for radionuclide content in sediments collected offshore of Bikini Atoll (IAEA-410) and in the open northwest Pacific Ocean (IAEA-412) are described and the results of the certification process are presented. The certified radionuclides include: (40)K, (210)Pb ((210)Po), (226)Ra, (228)Ra, (228)Th, (232)Th, (234)U, (238)U, (239)Pu, (239+240)Pu and (241)Am for IAEA-410 and (40)K, (137)Cs, (210)Pb ((210)Po), (226)Ra, (228)Ra, (228)Th, (232)Th, (235)U, (238)U, (239)Pu, (240)Pu and (239+240)Pu for IAEA-412. The CRMs can be used for quality assurance and quality control purposes in the analysis of radionuclides in sediments, for development and validation of analytical methods and for staff training.

S-myotrophin promotes the hypertrophy of myotube as insulin-like growth factor-I does.

We have reported previously that a novel muscle cell growth factor, having a structure of a peptide with sugar chains, was successfully purified from porcine skeletal muscle. It was named s-myotrophin. To determine the role of s-myotrophin in skeletal muscle growth, the effect of s-myotrophin on primary cultured chick skeletal muscle cells (composed almost totally of multinucleated myotubes) was investigated by comparing s-myotrophin with Insulin-like growth factor-I (IGF-I). Both s-myotrophin and IGF-I significantly increased creatine kinase activity of the cultures; both substances gave similar responses. Intracellar protein content was also increased by the addition of these factors. The content of myosin and actin in s-myotrophin treated culture in the differentiation medium was significantly higher than that of the control (unstimulated). The content of those proteins in IGF-I treated culture was also higher than that of control, but the differences were not statistically significant. Immunoblot analysis confirmed that the amounts of myosin and actin in the myocytes were greatly increased by s-myotrophin stimulation and also by IGF-I stimulation. Morphological observations using an anti-desmin antibody staining procedure demonstrated that the size of both s-myotrophin and IGF-I treated myotubes was appreciably larger than that of control myotubes. These results suggest that s-myotrophin is a potent mediator of skeletal muscle cell hypertrophy thorough the accumulations of muscle structural proteins.

Adenosine evokes potassium currents by protein kinase C activated via a novel signaling pathway in superior colliculus neurons.

Adenosine evoked whole-cell potassium currents and enhanced intracellular free Ca2+ concentration ([Ca2+]i) in superior colliculus neurons through a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein, possibly Gq-protein, which is involved in a protein kinase C (PKC) activation pathway. The [Ca2+]i increase was inhibited by a phospholipase C (PLC) inhibitor, whereas the evoked currents were not affected by a PLC inhibitor or a phospholipase A2 (PLA2) inhibitor. Adenosine elicited single channel currents via PKC activation in cell-attached patches and furthermore, those currents with conductances of the same slope were induced even in excised patches, suggesting that PKC can be activated only by cell membrane factors without intracellular components. These results thus indicate that the P2Y purinoceptor-coupled potassium channel is regulated via a novel PKC activation pathway independent of PLC or PLA2.

The enhancing of a cysteine proteinase activity at acidic pH by protein engineering, the role of glutamic 50 in the enzyme mechanism of caricain.

Carica papaya produces four cysteine proteinases. Calculations show that the Cys25, His159 essential ion pair is fully ionised at pH 2.99, where activity cannot be detected, but apparently an additional ionisation with a pKa of 4 is essential for activity (an electrostatic switch). Caricain (EC 3.4.22.30) wt and D158E genetic backgrounds were used to study the contribution of E50A to activity. E50 or E135 are candidates for the switch, E50A would be expected to reduce activity. However, activity increased at pH 5.0 in both backgrounds and at the pH optimum in D158E E50A but decreased slightly in the wt background. This challenges the hypothesis of an electrostatic switch.

Long-lasting enhancement of ACh receptor currents by lysophospholipids.

Lysophosphatidylcholine (LysoPtdCho) and lysophosphatidylethanolamine (LysoPtdEtn), which are formed by phospholipase A2-catalyzed hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), respectively, are proposed to be involved in protein kinase C (PKC) activation. Their physiological significance, however, remains unclear. We examined the effects of lysoPtdCho and lysoPtdEtn on acetylcholine (ACh) receptor currents using oocytes expressing Torpedo nicotinic ACh receptors. LysoPtdCho enhanced the currents in a washing time- and dose-dependent manner (10 nM-1 microM), reaching a maximum of 191% at 20 min after treatment. The currents were enhanced to a lesser extent at higher concentrations, and instead, inhibited to 81% at 10 microM. Likewise, lysoPtdEtn also potentiated the currents to 200% at 10 microM, although its dose-dependent curve shifted to right as compared with that of lysoPtdCho. The current potentiation was blocked by a PKC inhibitor, PKC inhibitor peptide (PKCI), or removal of extracellular Ca2+. In addition, lysoPtdCho and lysoPtdEtn enhanced the currents in mutant ACh receptors lacking PKC phosphorylation sites on the alpha and delta subunits. These results suggest that lysophospholipids such as lysoPtdCho and lysoPtdEtn potentiated ACh receptor currents by Ca2+-dependent PKC activation, but that this effect did not require PKC phosphorylation of the ACh receptor.

Effect of high hydrostatic pressure on the conversion of alpha-connectin to beta-connectin.

The factors affecting the conversion of alpha-connectin to beta-connectin induced by pressurization of muscle were investigated over a pressure range from 100 to 400 MPa by using SDS-PAGE and immunoblot analysis. When muscles were exposed to high pressures, the conversion of alpha-connectin to beta-connectin was the most pronounced at a pressure of 300 MPa, and the appearance of 1,200-kDa peptide accompanied by conversion of alpha- to beta-connectin was observed. Connectin was relatively resistant to degradation under a pressure of 400 MPa. The degradative products of beta-connectin reactive with mAb 2D4 were not observed. The effect of high pressure on connectin in isolated myofibrils was similar to that on connectin in muscle. Addition of leupeptin and E-64 to the isolated myofibrils resulted in the prevention of the degradation of connectin at each stage of the pressurization. The ability of calcium-activated protease (calpain) to hydrolyze connectin from alpha to beta gradually declined with increasing pressure. The results indicate that calpain is responsible for the pressure-induced conversion of alpha- to beta-connectin. The rate of this conversion is probably regulated by the pressure-dependent structural change of alpha-connectin and inactivation of calpain.

Instability of F-actin in the absence of ATP: a small amount of myosin destabilizes F-actin.

The effects of the neutral salt concentration, pH, and coexistence of myosin on the denaturation of F-actin without ATP at low temperature were studied using the DNase I inhibition assay. The percent denaturation of F-actin gradually increased with a decrease in pH from 8.0 to 5.2, on incubation for 2 weeks in the presence of 50 mM KCl at 0 degrees C. This change was much faster in 0.5 M KCl and more than 75% of the F-actin became denatured on incubation for 1 week at pH 5.2. The buffer composition was found to exert a strong influence on the denaturation of F-actin. That is, there was a tendency for the denaturation of F-actin at pH 6.0 to be faster in MES[2-(N-morpholino)ethanesulfonic acid]-NaOH buffer than in sodium phosphate buffer, the critical concentrations of actin in 0.5 M KCl being 0.31 mg/ml for MES-NaOH buffer and 0.15 mg/ml for sodium phosphate buffer. A sigmoidal relationship was found between the percent denaturation of F-actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.25 and 0.75 M. The time courses of the denaturation of F-actin showed that the percent denaturation rose at first and that in time the rate of the increase decreased. In the case of pH 8.0 and 0.5 M KCl, it took about 1 week for the denaturation rate to begin to drop. The pH of 6.0 further promoted the instability of F-actin exposed to high KCl concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Oleic acid enhances ACh receptor currents by activation of Ca2+/calmodulin-dependent protein kinase II.

Oleic acid, a cis-unsaturated free fatty acid, is proposed to be involved in the protein kinase C (PKC) activation pathway. Its biological actions, however, have not been well-characterized. We examined the effects of oleic acid on acetylcholine (ACh)-gated channel currents using Torpedo nicotinic ACh receptors expressed in Xenopus oocytes. Oleic acid (10 microM) enhanced the currents, reaching a maximum (140%) 20 min after treatment, while no enhancement was observed in Ca(2+)-free extracellular solution. The current potentiation by oleic acid was not inhibited by PKC inhibitors such as PKCI or GF109203X. Furthermore, oleic acid potentiated the currents in mutant ACh receptors lacking potential PKC phosphorylation sites. In contrast, the potentiation was fully inhibited by a CaMKII inhibitor, KN-62. These results strongly suggest that oleic acid potentiates ACh receptor currents by activation of calmodulin-dependent protein kinase II (CaMKII), independent of the PKC pathway.

A modified microsyringe for extracellular recording of neuronal activity.

We describe a modified Hamilton microsyringe that allows extracellular recording of neuronal activity and subsequent injections. It is assembled with a Hamilton removable needle and a syringe for injection, a Teflon-coated tungsten wire for recording, and polyimide tubing as a sheath. The device is inexpensive and easy to handle in anatomical and physiological experiments in awake monkeys.

Activation of endogenous protein kinase C enhances currents through alpha 1 and alpha 2 glycine receptor channels.

The effects of Ca2+ /phospholipid dependent (PKC) phosphorylation on the current amplitudes of alpha 1 and alpha 2 glycine receptors expressed in Xenopus oocytes were examined by whole cell voltage clamp recording. In studies using phorbol esters, PKC phosphorylation has been shown to reduce glycine-induced currents. Endogenous PKC activation by pretreatment with serum, however, enhanced the currents to around 140% in both alpha 1 and alpha 2 glycine receptors. This effect was completely blocked by a specific PKC inhibitor, GF109203X. Instead, treatment with a potent PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) revealed a decrease in glycine-gated channel currents. Thus, the present results demonstrate that glycine receptor phosphorylation mediated by endogenous pathway of PKC activation potentiates glycine-induced currents and phorbol esters may have a direct action on glycine receptor channels independent of PKC activation.

Toluene inhibits synaptic transmission without causing gross morphological disturbances.

The effects of toluene on synaptic transmission and neuronal morphology were investigated using guinea pig hippocampal slices. Population spikes (PS) were elicited in granule cell layer by stimulation of the perforant path and antidromic potentials (AP) were evoked in the same locii by stimulation of mossy fibers. Toluene at a concentration of 1000 micrograms/ml completely depressed post-synaptic responses within 15 min but increased the AP to 140% of its original value. The PS recovered completely when washout was begun 10 and 20 min after onset of depression, but exhibited only partial recovery following a 40 min depression. There were no evident changes in staining of axons or cell bodies after toluene treatment. These results indicate that toluene at high concentrations (1000 micrograms/ml) inhibits synaptic transmission selectively and with longer exposures causes lasting physiological effects unaccompanied by gross morphological changes.

Short-term depression and long-term enhancement of ACh-gated channel currents induced by linoleic and linolenic acid.

The effects of cis-unsaturated free fatty acids such as linoleic and linolenic acid on ACh-evoked currents were examined using normal and mutant nicotinic acetylcholine (ACh) receptors lacking protein kinase C (PKC) phosphorylation sites on the alpha and delta subunits expressed in Xenopus oocytes. These free fatty acids reduced ACh-gated channel currents during treatment and to a greater extent in Ca2+-free extracellular solution. After treatment, the currents were enhanced as the drug was washed out, but this effect was not observed in the absence of extracellular Ca2+. Linolenic acid was more potent of the current enhancement (300% of the control) than linoleic acid (190% of the control). The current enhancement induced by these free fatty acids was inhibited by the selective PKC inhibitor, GF109203X, while the current depression was not affected. Furthermore, these lipids decreased ACh-evoked currents in mutant ACh receptors to the same extent as in normal ACh receptors, but never enhanced the currents. These results indicate that linoleic and linolenic acid have biphasic actions on ACh receptor currents; a short-term depression and a long-term enhancement. The short-term depression may be due to an interaction with the ACh receptor channels, presumably at Ca2+ binding sites. The long-lasting enhancement appears to result from Ca2+-dependent PKC activation followed by PKC phosphorylation of the ACh receptors.

ATP-regulated K+ channel and cytosolic Ca2+ mobilization in cultured rat spinal neurons.

ATP activated the K+ channel responsible for outwardly rectifying currents via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein in cultured rat spinal neurons. The evoked currents were inhibited by a selective protein kinase C inhibitor, GF109203X, whereas a phospholipase C inhibitor, neomycin had no effect. These indicate that the currents are regulated by phospholipase C-independent protein kinase C activation. In addition, ATP enhanced intracellular free Ca2+ concentration. The increase in intracellular free Ca2+ concentration was inhibited by a broad G-protein inhibitor, GDP beta S, but not affected by neomycin or an inositol 1,4,5-triphosphate receptor antagonist, heparin, suggesting that the cytosolic Ca2+ mobilization is regulated by a mechanism independent of a phospholipase C-mediated phosphatidylinositol signaling. These results, thus, demonstrate that ATP has dual actions on the coupled K+ channel and cytosolic Ca2+ release.

Adenosine activates the K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor in hippocampal neurons.

The effects of adenosine on hippocampal neurons were examined by patch-clamp recording and Ca2+ imaging using fura-2 fluorescence. In the whole-cell patch-clamp configuration, adenosine evoked outwardly rectifying K+ currents in a dose-dependent manner. These currents were not inhibited by a nonselective P1 purinoceptor antagonist or selective adenosine A1, A2A receptor antagonists and moreover, selective adenosine A1, A2A receptor agonists evoked no current. In contrast, P2 purinoceptor agonists produced similar outward currents with the order of potency: ADP > or = 2-methylthio ATP > ATP > adenosine >> AMP. No response was obtained to UTP, alpha, beta-methylene ATP or beta, gamma-methylene ATP. The intracellular perfusion of a broad G-protein inactivator, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), abolished adenosine-evoked currents, whereas a Gi/Go-protein inhibitor, pertussis toxin, had no effect. Furthermore, the currents were blocked by a phospholipase C inhibitor, neomycin, or specific protein kinase C inhibitors, GF109203X (bisindolyl maleimide, C25H24N4O2) and protein kinase C inhibitor peptide. In the cell-attached patch-clamp configuration, adenosine elicited single-channel currents with two major kinds of slope conductances. Likewise, application of adenosine outside the patch electrode again produced single-channel currents with same conductances. A potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced single-channel currents in a fashion that mimics the effect of adenosine. The evoked currents were blocked by GF109203X. In addition, adenosine enhanced intracellular free Ca2+ concentration ([Ca2+]i). This [Ca2+]i increase was inhibited by GDP beta S or neomycin, but was not affected by pertussis toxin. These results, thus, suggest that adenosine activates the K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein, which is involved in a phospholipase C-mediated phospholipid-signaling pathway.

Methylcobalamin induces a long-lasting enhancement of the postsynaptic field potential in hippocampal slices of the guinea pig.

Methylcobalamin (CH3-B12) enhanced postsynaptic field potential (PSP) elicited from hippocampal slices (around 180% at a maximum), lasting more than 1 h after washing out, while it had no effect on antidromic spike potential (AP). By contrast, cyanocobalamin containing CN- instead of CH3- showed no enhancement in the PSP amplitude, suggesting that CH3- plays an important role for the PSP enhancement by methylcobalamin. The treatment with N-methyl-D-aspartate (NMDA) receptor specific antagonist, DL-2-amino-5-phosphonovaleric acid (APV) inhibited methylcobalamin-induced PSP enhancement by 50%, suggesting that NMDA receptor is partially relevant to the formation of this enhancement. Glutamate release from electrically stimulated slices was not enhanced by the treatment with methylcobalamin, suggesting that methylcobalamin affects the postsynaptic sites in hippocampal neurons. In whole cell voltage clamp recordings using cultured hippocampal neurons, methylcobalamin increased the currents elicited by NMDA time-dependently. These results may indicate that CH3- is required to modulate the PSP amplitude at the postsynaptic site and at least, NMDA receptor is involved in the formation of long-lasting PSP potentiation induced by methylcobalamin.