Starvation induces tau hyperphosphorylation in mouse brain: implications for Alzheimer's disease.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease brains, and tau hyperphosphorylation is thought to be a critical event in the pathogenesis of this disease. The objective of this study was to reproduce tau hyperphosphorylation in an animal model by inducing hypoglycemia. Food deprivation of mice for 1 to 3 days progressively enhanced tau hyperphosphorylation in the hippocampus, to a lesser extent in the cerebral cortex, but the effect was least in the cerebellum, in correspondence with the regional selectivity of tauopathy in Alzheimer's disease.

Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity.

Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2, 4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated.

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa.

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant.

Lithium inhibits neurite growth and tau protein kinase I/glycogen synthase kinase-3beta-dependent phosphorylation of juvenile tau in cultured hippocampal neurons.

Tau protein kinase I(TPKI)/glycogen synthase kinase (GSK)-3beta is abundant in the developing rat brain. The highly phosphorylated juvenile form of tau is present during the same developmental period. To study the role of TPKI/ GSK-3beta in neuronal growth, we examined the effects of lithium, a direct inhibitor of TPKI/GSK-3beta, using primary cultures of rat hippocampal neurons.

An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence for novel substrate recognition by a cysteine protease.

Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the removal of ubiquitin C-terminal adducts, is important for the generation of monomeric ubiquitin. Heteronuclear NMR spectroscopy has been utilized to map the YUH1 binding surface on ubiquitin. When YUH1 was titrated into a sample of ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin were affected to some degree, as a result of binding to YUH1.

Mouse CAF1, a mouse homologue of the yeast POP2 gene, complements the yeast pop2 null mutation.

The yeast POP2 protein (Pop2p) is a component of a global transcription regulatory complex and is required for gene expression of many genes in Saccharomyces cerevisiae. We constructed POP2 deletion plasmids encoding various Pop2p regions under the native POP2 promoter and found that the minimum functional region was located in two-thirds of the carboxyl terminal region. A mouse homologue of the POP2 gene (mCAF1), which corresponds to the Pop2p minimum region, partially rescued the growth defect of pop2 null mutant cells.

Synthesis, bioactivity, and cloning of the L-type calcium channel blocker omega-conotoxin TxVII.

omega-Conotoxin TxVII is the first conotoxin reported to block L-type currents. In contrast to other omega-conotoxins, its sequence is characterized by net negative charge and high hydrophobicity, although it retains the omega-conotoxin cysteine framework. In order to obtain structural information and to supply material for further characterization of its biological function, we synthesized TxVII and determined its disulfide bond pairings.

Early postnatal development of substantia nigra neurons in rat midbrain slices: hyperpolarization-activated inward current and dopamine-activated current.

Whole-cell voltage-clamp recordings in rat midbrain slices were used to determine the characteristics and postnatal development of a hyperpolarization-activated inward current (Ih) and a dopamine receptor-activated current of neurons in the substantia nigra pars compacta (SNC). Ih was activated by membrane hyperpolarization beyond the resting membrane potentials, and displayed strong inward rectification. No sign of time- and voltage-dependent inactivation was apparent.

Corticocortical associative neurons expressing latexin: specific cortical connectivity formed in vivo and in vitro.

Latexin, a carboxypeptidase A inhibitor, is expressed in a subset of neurons in the infragranular layers of the lateral cortex in the rat. We here show that latexin-expressing neurons exhibit ultrastructural features common to cortical pyramidal neurons. We show in combined retrograde tracing and immunofluorescent experiments that latexin-expressing neurons contribute to specific corticocortical pathways.

Insertional gene fusion technology.

The classical 'end to end' gene fusion technique has widely been used for monitoring gene expression, biological screening and purification of recombinant proteins. Recent progress with the 'insertional' gene fusion approach, on the other hand, has demonstrated that this technique can be utilized for membrane protein topology analysis, display of randomized protein libraries and design of biosensor proteins. In this review, we describe examples of insertional gene fusion and compare the old and new gene fusion techniques.

STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro.

We have developed a new method that permits the complete in vitro construction and selection of peptide or protein libraries. This method relies on an in vitro transcription/translation reaction compartmentalized in water in oil emulsions. In each emulsion compartment, streptavidin (STA)-fused polypeptides are synthesized and attached to the encoding DNA via its biotin label.

Phosphorylated tau in human cerebrospinal fluid is a diagnostic marker for Alzheimer's disease.

Microtubule-associated protein tau in cerebrospinal fluid (CSF) has been proposed as a diagnostic marker for Alzheimer's disease (AD), but there is overlap between AD patients and non-AD controls. To improve the diagnostic accuracy, we measured phosphorylated tau in CSF, because phosphorylated tau accumulates as pathological paired helical filaments in neurons of the AD brain. Immunoblot showed that CSF contained a 32 kDa N-terminal fragment of tau that was partially phosphorylated on Ser199, Thr231 and Ser235.

The organization of extrinsic neurons and their implications in the functional roles of the mushroom bodies in Drosophila melanogaster Meigen.

Although the importance of the Drosophila mushroom body in olfactory learning and memory has been stressed, virtually nothing is known about the brain regions to which it is connected. Using Golgi and GAL4-UAS techniques, we performed the first systematic attempt to reveal the anatomy of its extrinsic neurons. A novel presynaptic reporter construct, UAS-neuronal synaptobrevin-green fluorescent protein (n-syb-GFP), was used to reveal the direction of information in the GAL4-labeled neurons.

jumonji gene is essential for the neurulation and cardiac development of mouse embryos with a C3H/He background.

The recessive mutant mouse jumonji (jmj), obtained by a gene trap strategy, shows neural tube defects in approximately half of homozygous embryos with a BALB/cA and 129/Ola mixed background, but no neural tube defects with BALB/cA, C57BL/6J, and DBA/2J backgrounds. Here, we show that neural tube and cardiac defects are observed in all embryos with a C3H/HeJ background. In addition, abnormal groove formation and prominent flexure are observed on the neural plate with full penetrance, suggesting that abnormal groove formation leads to neural tube defects.

Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation.

A high level of foreign gene expression in organotypic cultures of the cerebral cortical anlage was achieved by electroporation-mediated gene transfer in vivo. A mammalian expression plasmid for green fluorescent protein (GFP) gene was injected into the lateral ventricle of rat embryos. Immediately after the plasmid DNA injection, the head of the embryo was electroporated between a pair of tweezer-type electrodes.

Increased anxiety and impaired pain response in puromycin-sensitive aminopeptidase gene-deficient mice obtained by a mouse gene-trap method.

A mouse mutation, termed goku, was generated by a gene-trap strategy. goku homozygous mice showed dwarfism, a marked increase in anxiety, and an analgesic effect. Molecular analysis indicated that the mutated gene encodes a puromycin-sensitive aminopeptidase (Psa; EC 3.

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.

Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs.

Mouse gene trap approach: identification of novel genes and characterization of their biological functions.

The mouse gene trap strategy is an insertional mutagenesis involving an exogenous DNA, termed the trap vector, as a mutagen that produces a mutation in the mouse genome and a sequence tag to facilitate the isolation of the mutated genes. The trap vector consists of a reporter gene whose expression mimics that of the endogenous genes mutated and a selection marker that sorts cells bearing the inserted vector. Gene trap is a powerful method for identifying genes important in biological phenomena.

Severe impairment of B cell function in lpr/lpr mice expressing transgenic Fas selectively on B cells.

Transgenic lpr/lpr mice expressing functional Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene did not produce anti-double-stranded DNA antibodies throughout their lives, whereas the development of abnormal lpr T cells (double negative, B220(+)) was not suppressed. Further analyses, however, revealed that the expression of the transgenic Fas on B cells of lpr/lpr homozygous mice resulted in severe impairment of the B cell function.

Monoaminergic long-term facilitation of GABA-mediated inhibitory transmission at cerebellar synapses.

Long-term facilitation of neurotransmission by monoaminergic systems is implicated in the cellular mechanism of memory and learning-related processes at invertebrate synapses. Using whole-cell recording and rat cerebellar slices, we have examined whether mammalian monoamine-containing neurons play analogous roles in synaptic plasticity, and our results suggest that serotonin and noradrenaline are critically involved in short- and long-term modulation of GABAergic transmission in the cerebellar cortex. Exogenously applied serotonin and noradrenaline selectively induced a short-term enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells, their effect subsiding in 30 min.

Partial nuclear localization of a bovine phosphoprotein, BCNT, that includes a region derived from a LINE repetitive sequence in Ruminantia.

BCNT, named after Bucentaur, is a protein that contains a 324-amino-acid region derived from part of a long interspersed DNA sequence element (LINE) in Ruminantia. However, the unique portion is completely missing in human and mouse BCNTs. Since no significant information on their function has been obtained by homology search, we at first examined cellular localization and biochemical characteristics of bovine BCNT to get a hint on its function.

Genomic organization and regulatory elements of the rat latexin gene, which is expressed in a cell type-specific manner in both central and peripheral nervous systems.

Latexin, a carboxypeptidase A inhibitor, is expressed in a cell type-specific manner in both central and peripheral nervous systems in the rat. In the neocortex, a specific subpopulation of neurons in layers V and VI expresses latexin. In the primary sensory ganglia, the expression is restricted to smaller diameter neurons.

Expression of the spermatid-specific Hsp70 antigen is conserved in mammals including marsupials.

The anatomical location of testes in mammals ranges from a location close to that observed in the embryo to a lower position usually involving a pendant scrotum. In scrotal mammals, the abdominal position of the cryptorchid testis, which elevates its temperature, is detrimental to spermatogenesis and causes infertility. Spermatocytes are sensitive but late spermatids are relatively resistant to thermal stress suggesting that the latter might be protected in some way.