Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome.

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells.

Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.

Chronic Hyponatremia Causes Neurologic and Psychologic Impairments.

Hyponatremia is the most common clinical electrolyte disorder. Once thought to be asymptomatic in response to adaptation by the brain, recent evidence suggests that chronic hyponatremia may be linked to attention deficits, gait disturbances, risk of falls, and cognitive impairments. Such neurologic defects are associated with a reduction in quality of life and may be a significant cause of mortality. However, because underlying diseases such as adrenal insufficiency, heart failure, liver cirrhosis, and cancer may also affect brain function, the contribution of hyponatremia alone to neurologic manifestations and the underlying mechanisms remain unclear. Using a syndrome of inappropriate secretion of antidiuretic hormone rat model, we show here that sustained reduction of serum sodium ion concentration induced gait disturbances; facilitated the extinction of a contextual fear memory; caused cognitive impairment in a novel object recognition test; and impaired long-term potentiation at hippocampal CA3-CA1 synapses. In vivo microdialysis revealed an elevated extracellular glutamate concentration in the hippocampus of chronically hyponatremic rats. A sustained low extracellular sodium ion concentration also decreased glutamate uptake by primary astrocyte cultures, suggesting an underlying mechanism of impaired long-term potentiation. Furthermore, gait and memory performances of corrected hyponatremic rats were equivalent to those of control rats. Thus, these results suggest chronic hyponatremia in humans may cause gait disturbance and cognitive impairment, but these abnormalities are reversible and careful correction of this condition may improve quality of life and reduce mortality.

BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm.

During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES) cells. Generally, any lot differences in fetal bovine serum (FBS) and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9-36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP) 4 and fibroblast growth factor (FGF) treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78-90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells.

Rabphilin-3A as a Targeted Autoantigen in Lymphocytic Infundibulo-neurohypophysitis.

CONTEXT: Central diabetes insipidus (CDI) can be caused by several diseases, but in about half of the patients the etiological diagnosis remains unknown. Lymphocytic infundibulo-neurohypophysitis (LINH) is an increasingly recognized entity among cases of idiopathic CDI; however, the differential diagnosis from other pituitary diseases including tumors can be difficult because of similar clinical and radiological manifestations. The definite diagnosis of LINH requires invasive pituitary biopsy. OBJECTIVE: The study was designed to identify the autoantigen(s) in LINH and thus develop a diagnostic test based on serum autoantibodies. DESIGN: Rat posterior pituitary lysate was immunoprecipitated with IgGs purified from the sera of patients with LINH or control subjects. The immunoprecipitates were subjected to liquid chromatography-tandem mass spectrometry to screen for pituitary autoantigens of LINH. Subsequently, we made recombinant proteins of candidate autoantigens and analyzed autoantibodies in serum by Western blotting. RESULTS: Rabphilin-3A proved to be the most diagnostically useful autoantigen. Anti-rabphilin-3A antibodies were detected in 22 of the 29 (76%) patients (including 4 of the 4 biopsy-proven samples) with LINH and 2 of 18 (11.1%) patients with biopsy-proven lymphocytic adeno-hypophysitis. In contrast, these antibodies were absent in patients with biopsy-proven sellar/suprasellar masses without lymphocytic hypophysitis (n = 34), including 18 patients with CDI. Rabphilin-3A was expressed in posterior pituitary and hypothalamic vasopressin neurons but not anterior pituitary. CONCLUSIONS: These results suggest that rabphilin-3A is a major autoantigen in LINH. Autoantibodies to rabphilin-3A may serve as a biomarker for the diagnosis of LINH and be useful for the differential diagnosis in patients with CDI.

TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome.

Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease-mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome.

CREB-binding protein transcription activation domain for enhanced transgene expression by a positive feedback system.

The positive feedback system using a fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the transcription activation domain of herpes simplex virus VP16 (GAL4-VP16), in which GAL4-VP16 promotes its own expression as well as that of a reporter gene product, is useful for efficient transgene expression from plasmid DNA. In this study, the transcription activation domains of endogenous proteins, instead of VP16, were fused to the GAL4 DNA binding domain, and the positive feedback systems employing the novel fusion proteins were examined. Plasmid DNAs encoding the transcription factors were introduced into mouse Hepa 1-6 cells by electroporation and lipofection. Among CREB-binding protein (226-460), sterol regulatory element-binding protein-1 (1-140), p53 (1-70), and Med15 (9-73), the CREB-binding protein functioned efficiently as an activator. These results indicated that the GAL4-CREB-binding protein is useful for enhanced transgene expression by the positive feedback system.

Protein kinase B gene homologue pkbR1 performs one of its roles at first finger stage of Dictyostelium.

Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene (pkaC) only in the prestalk region of the pkbR1 null mutant.

Fulminant type 1 diabetes mellitus associated with a reactivation of Epstein-Barr virus that developed in the course of chemotherapy of multiple myeloma.

A 70-year-old woman who was diagnosed with multiple myeloma underwent chemotherapy. Three months after beginning chemotherapy, she was readmitted to the hospital because of fever and hepatopathy. Her elevated Epstein-Barr virus (EBV) antibody levels showed that the hepatopathy was caused by reactivation of EBV. On the 18th hospital day, the levels of fasting plasma glucose (FPG; 451 mg/dL) and pancreatic enzymes were suddenly elevated. Elevation of HbA1c level (6.4%) was slight, as compared with that of the FPG level. Arterial blood gas analysis showed metabolic acidosis and diabetic ketoacidosis was suspected. The serum C-peptide level was below the detectable limit both before and after glucagon load, thereby suggesting an insulin-dependent state. These features were identical to the features for fulminant type 1 diabetes mellitus. The levels of EBV anti-viral capsid antigen immunoglobulin M decreased, and the clinical course was identical to that associated with reactivation of EBV infection. (J Diabetes Invest, doi: 10.1111/j.2040.1124.2010.00061.x, 2010).

Role of the nanos homolog during sea urchin development.

The nanos genes play important roles in the development of primordial germ cells in animal species. In the sea urchin, Hemicentrotus pulcherrimus, small micromere descendants specifically express HpNanos mRNA and this expression continues in the left coelomic pouch, which produces the major component of the adult rudiment. In this study, we showed that morpholino knockdown of HpNanos resulted in a delay of primary mesenchyme cell ingression and a decrease in the number of cells comprising the left coelomic pouch. Knockdown analysis in chimeras and whole embryos revealed the disappearance of small micromere descendants from the archenteron tip. Furthermore, the expression of HpNanos mRNA was induced in other cell lineages in the HpNanos-knockdown and micromere-deleted embryos. Taken together, our results suggest that HpNanos is involved in the inductive interaction of small micromere descendants with other cell lineages, and that HpNanos is required for the survival of small micromere descendants.

Inhibitory Effect of TNF-alpha Produced by Macrophages Stimulated with Grifola frondosa Extract (ME) on the Growth of Influenza A/Aichi/2/68 Virus in MDCK Cells.

We investigated the inhibitory effect of the conditioned medium (CM) from P338D1 (D1) cells, a murine macrophage cell line, stimulated for 10 hours with a fixed dose (100 mug/ml) of the extracts from the fruit bodies of Grifola frondosa (ME) or its ultra filtration-based fractions (MFs), on the growth of influenza A/Aichi/2/68 virus in Madin-Darby canine kidney cells. Direct addition of ME and 3 kinds of MFs (MF1, MF2 and MF3) to the infected cells had no obvious inhibitory effect. However, virus yields were reduced in the presence of CMs. Notably, the inhibitory effect of the CM prepared by using MF2 (molecular weight of 30 Kd to 100 Kd) was the strongest (28% reduction compared to the control). RT-PCR and ELISA assays showed that the CMs could induce the expression of TNF-alpha mRNA in D1 cells leading to production of TNF-alpha, known as an antiviral cytokine. These findings suggest that ME and MFs (especially MF-2) might induce the production of certain factors, including TNF-alpha, which are responsible for the inhibition of viral growth in vitro.

Successful treatment of chronic granulomatous disease with fludarabine-based reduced-intensity conditioning and unrelated bone marrow transplantation.

Allogeneic hematopoietic stem-cell transplantation (HSCT) for chronic granulomatous disease (CGD) with a reduced-intensity conditioning regimen can be expected to lead to less therapy-related mortality and late-onset impairment, whereas it has also been reported to increase the risk of unsustained mixed donor chimerism and late rejection after transplantation. Herein, we report a 4-year-old boy with CGD who was successfully treated with unrelated bone marrow transplantation with a reduced-intensity conditioning regimen (RIC). Fludarabine-based RIC, 4 Gy of total body irradiation, 120 mg/kg of cyclophosphamide, and 125 mg/m(2) of fludarabine, was adopted for transplantation, followed with 8.9 x 10(8)/kg mononucleated donor cells infused without T-cell depletion. Although hematopoietic engraftment was rapidly obtained by day +17, he developed unstable donor chimerism. After tacrolimus withdrawal, the patient showed grade III acute graft-versus-host disease (GVHD), and subsequently reached full donor chimerism by day +61. Twelve months post-transplant, the patient has remained well with stable and durable engraftment, 100% donor chimerism, and normal superoxide production, without the requirement of donor lymphocyte infusions (DLI).

Chotosan enhances macrophage colony-stimulating factor mRNA expression in the ischemic rat brain and C6Bu-1 glioma cells.

Macrophage colony stimulating factor (M-CSF) is a cytokine which has been recently reported to have a neuroprotective effect on ischemic rat brain. In this study, we investigated the effect of chotosan, an oriental medicine, which has been clinically demonstrated to be effective for the treatment of vascular dementia, on M-CSF gene expression in rats with permanent occlusion of bilateral common carotid arteries (P2VO) in vivo and in a C6Bu-1 glioma cell line in vitro. The expression level of M-CSF mRNA in the cerebral cortices of P2VO rats was significantly higher than that in the cerebral cortices of sham-operated animals. Repeated treatment of P2VO rats with chotosan (75 mg/kg per day) for 4 d after P2VO significantly increased the expression level of M-CSF mRNA in the cortex but it had no effect on the expression of beta-actin, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF) mRNAs. Moreover, the present in vitro studies revealed that chotosan treatment (10-100 mug/ml) of C6Bu-1 glioma cells dose-dependently enhanced M-CSF mRNA expression without affecting the expression of G-CSF, GM-CSF, and inducible nitric oxide synthase mRNAs. The effect of chotosan was reversed by Ro 31-8220 (1 muM), a selective protein kinase C (PKC) inhibitor, but not by H-89 (10 muM), a selective protein kinase A (PKA) inhibitor. These findings suggest that the upregulatory effect of chotosan on M-CSF mRNA expression involves PKC and may play an important role in the anti-vascular dementia action of this formula.

Enzymatic and biochemical properties of a novel human serine dehydratase isoform.

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

Macrophage-mediated inhibitory effect of Zingiber officinale Rosc, a traditional oriental herbal medicine, on the growth of influenza A/Aichi/2/68 virus.

The inhibitory effect of Zingiber officinale Rosc (ZOR), an Oriental traditional herbal medicine, on the growth of influenza A/Aichi/2/68 (Aichi) virus was investigated in Madin-Darby canine kidney (MDCK) cells. Direct addition of ZOR (0.1 approximately 100 microg/ml) to the infected cells did not have any inhibitory effect. However, the ZOR-induced conditioned medium (ZOR-CM) of RAW cells, a murine macrophage (Mphi) cell line, exhibited an apparent inhibitory effect on MDCK cells without cytotoxicity. In accordance with the time-dependent inhibitory effect of ZOR-CM, it has been demonstrated that tumor necrosis factor (TNF)-alpha was gradually accumulated in ZOR-CM by the induction of TNF-alpha mRNA expression in ZOR-stimulated RAW cells. Conversely, the inhibitory effect of ZOR-CM was reduced significantly by the removal of TNF-alpha after the formation of an immune complex with anti-TNF-alpha monoclonal antibody. These data suggested that ZOR itself has no inhibitory effect on the growth of influenza virus, but could exert its effect via macrophage activation leading to production of TNF-alpha.

Induction of inducible nitric oxide (NO) synthase mRNA and NO production in macrophages infected with influenza A/PR/8 virus and stimulated with its ether-split product.

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether-split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (M psi) cell line, and thioglycolate-elicited peritoneal M psi (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS-mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.

Design, synthesis, and biological evaluation of new phosphodiesterase type 4 inhibitors.

The design, synthesis, and biological evaluation of new phosphodiesterase type 4 inhibitors, which possess new templates instead of a cyclohexane ring, are described. The mode of interaction with the enzyme is discussed based on the structure-activity relationship (SAR) data obtained for the synthesized inhibitors. Furthermore, the roles of three pharmacophores, a catechol moiety, a nitrile moiety, and acidic moieties, are discussed using in silico docking studies. More detailed biological evaluations of selected compounds are also presented.

Discovery of new orally active phosphodiesterase (PDE4) inhibitors.

A series of 4-anilinopyrazolopyridine derivatives were synthesized and biologically evaluated as inhibitors of phosphodiesterase (PDE4). Chemical modification of 3, a structurally new chemical lead that was found in our in-house library, was focused on 1- and 3-substituents. Full details of the discovery of a new orally active chemical lead 5 are presented. Structure-activity relationship data, pharmacological evaluation, and the subtype selectivity study are also presented.

Orally active PDE4 inhibitor with therapeutic potential.

Based on the promising results obtained by the clinical trial of Ariflo, further optimization of the spatial arrangement of the three pharmacophores (the carboxylic acid moiety, nitrile moiety and 3-cyclopentyloxy-4-methoxyphenyl moiety) in the structure of Ariflo 1 was attempted using a bicyclo[3 ?3 ?0]octane template with more stereochemical diversity than the cyclohexane template of Ariflo 1. Biological evaluation of the decyanated analogs and further optimization of the cyclopentyloxy moiety of 2a-b were also performed. Among the compounds tested, 2a, 7a-b and 12a were found to be orally active and were estimated to have therapeutic potential based on cross-species and same-species comparisons. The structure-activity relationships (SARs) of these compounds were investigated and pharmacokinetic data for 2a and 7b were also obtained by single-dose studies in rats.