LRRK2 phosphorylates tubulin-associated tau but not the free molecule: LRRK2-mediated regulation of the tau-tubulin association and neurite outgrowth

Leucine-rich repeat kinase 2 (LRRK2), a large protein kinase containing multi-functional domains, has been identified as the causal molecule for autosomal-dominant Parkinson's disease (PD). In the present study, we demonstrated for the first time that (i) LRRK2 interacts with tau in a tubulin-dependent manner; (ii) LRRK2 directly phosphorylates tubulin-associated tau, but not free tau; (iii) LRRK2 phosphorylates tau at Thr181 as one of the target sites; and (iv) The PD-associated LRRK2 mutations, G2019S and I2020T, elevated the degree of tau-phosphorylation. These results provide direct proof that tau is a physiological substrate for LRRK2. Furthermore, we revealed that LRRK2-mediated phosphorylation of tau reduces its tubulin-binding ability. Our results suggest that LRRK2 plays an important role as a physiological regulator for phosphorylation-mediated dissociation of tau from microtubules, which is an integral aspect of microtubule dynamics essential for neurite outgrowth and axonal transport.     

ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.     

Identification and characterization of the cis-elements regulating the rat AMOG (adhesion molecule on glia)/Na,K-ATPase beta 2 subunit gene.

We have identified a positive regulatory cis-acting element of the adhesion molecule on glia (AMOG)/Na,K-ATPase beta 2 subunit gene as GAGGCGGGG at position -87 to -79 by transient transfection assay using B103 cells (rat neuroblastoma cell line). The newly identified AMOG regulatory element (AMRE) enhanced the promoter activity in a mutually compensating manner with the Sp1 element at position -147 to -142. AMRE acts as a positive regulatory element not only in B103 cells but also in 3Y1 (rat embryo cell line) cells to roughly the same extent and in MDCK (canine kidney cell line) cells to a lesser extent. AMRE also enhances other gene promoters, such as myelin basic protein (MBP) and herpes simplex virus (HSV) thymidine kinase (TK) gene promoters. The element is not a typical enhancer element because when it is introduced downstream of the HSV TK promoter, it has no enhancing activity.     

Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand–receptor combinations that occur concurrently during development in zebrafish.     

Risk Factors for Cisplatin-Induced Nephrotoxicity and Potential of Magnesium Supplementation for Renal Protection

Background

Nephrotoxicity remains a problem for patients who receive cisplatin chemotherapy. We retrospectively evaluated potential risk factors for cisplatin-induced nephrotoxicity as well as the potential impact of intravenous magnesium supplementation on such toxicity.

Patients and Methods

We reviewed clinical data for 401 patients who underwent chemotherapy including a high dose (≥60 mg/m²) of cisplatin in the first-line setting. Nephrotoxicity was defined as an increase in the serum creatinine concentration of at least grade 2 during the first course of cisplatin chemotherapy, as assessed on the basis of National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. The severity of nephrotoxicity was evaluated on the basis of the mean change in the serum creatinine level. Magnesium was administered intravenously to 67 patients (17%).

Results

Cisplatin-induced nephrotoxicity was observed in 127 patients (32%). Multivariable analysis revealed that an Eastern Cooperative Oncology Group performance status of 2 (risk ratio, 1.876; P = 0.004) and the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) (risk ratio, 1.357; P = 0.047) were significantly associated with an increased risk for cisplatin nephrotoxicity, whereas intravenous magnesium supplementation was associated with a significantly reduced risk for such toxicity (risk ratio, 0.175; P = 0.0004). The development of hypomagnesemia during cisplatin treatment was significantly associated with a greater increase in serum creatinine level (P = 0.0025). Magnesium supplementation therapy was also associated with a significantly reduced severity of renal toxicity (P = 0.012).

Conclusions

A relatively poor performance status and the regular use of NSAIDs were significantly associated with cisplatin-induced nephrotoxicity, although the latter association was marginal. Our findings also suggest that the ability of magnesium supplementation to protect against the renal toxicity of cisplatin warrants further investigation in a prospective trial.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies

ObjectiveAutoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies.MethodsHere we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients’ serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated.ResultsIn patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006).ConclusionOur newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM.     

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.     

Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by¹H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (P^k-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tj^a serum is the placenta tissue, resulting in termination of the pregnancy.