Measurement of endogenous leucine enkephalin in canine caudate nuclei and hypothalami with high-performance liquid chromatography and field-desorption mass spectrometry

For the first time, endogenous amounts of Leu-enkephalin are measured in brain tissue with a technique preserving integrity of the entire molecular structure of the neuropeptide. Field-desorption mass spectrometry enables measurement of picomole amounts of endogenous, chemically underivatized Leu-enkephalin in canine caudate nuclei and hypothalami. The optimal sensitivity and resolution of high-performance liquid chromatography is coupled with maximal molecular specificity of field-desorption mass spectrometry to measure enkephalins in caudate nuclei and hypothalami from dog brains. This novel combination of two recent instrumental methodologies provides a firm molecular basis for calibrating the radioimmunoassay measurement of endogenous levels of biologically active brain neuropeptides.     

A novel mannose-specific and sugar specifically aggregatable lectin from the bark of the Japanese pagoda tree (Sophora japonica)

A new D-mannose/D-glucose-specific lectin (B-SJA-II) was isolated from the bark of the Japanese pagoda tree, Sophora japonica. B-SJA-II was separated from a well known D-galactose/N-acetyl-D-galactosamine-specific lectin (B-SJA-I) by affinity chromatography on lactamyl-Sepharose, then purified by affinity chromatography on maltamyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B-SJA-II gave four bands: subunit a-1 (Mr = 19,400), a-2 (Mr = 18,200), b-1 (Mr = 15,000), and b-2 (Mr = 13,200). Carbohydrate analysis and binding study with horseradish peroxidase-labeled lectins on the bands electroblotted onto polyvinylidene difluoride membrane showed that the three subunits other than b-2 have N-linked oligosaccharides typical of plant glycoproteins. The binding assay with horseradish peroxidase-glycoproteins revealed that all the subunits can bind sugar specifically with fetuin and asialofetuin. Furthermore, B-SJA-II aggregated to form precipitates in the absence of a specific sugar and became soluble upon addition of the specific sugar. The results indicate that each subunit has a sugar-binding site for the mannosyl core of N-linked oligosaccharide chains and recognizes each other sugar specifically to form aggregates. According to the N-terminal amino acid sequences obtained, the subunits are classified into two groups. The first group (a-1 and a-2) has an N-terminal sequence 50% identical with that of other S. japonica lectins (Hankins, C. N., Kindinger, J. I., and Shannon, L. M. (1988) Plant Physiol. 86, 67-70) and the amino acid sequence initiating at position 123 of concanavalin A (Cunningham, B. (1975) J. Biol. Chem. 250, 1503-1512), while the N-terminal sequence of the second group (b-1 and b-2) is homologous to that of concanavalin A, but completely different from that of the first group.     

Increase in dATP pool in aphidicolin-resistant mutants of mouse FM3A cells

Mutants that were resistant to aphidicolin were isolated from mutagenized mouse FM3A cells at a frequency of about 10^?6. Resistance to aphidicolin in these mutants was not due to an effect on [³H]thymidine incorporation into DNA, DNA synthesis in permeabilized cells, or DNA polymerase α.All the mutants showed a greatly increased dATP pool and decreased ability to incorporate [³H]deoxycytidine into DNA. They also showed cross-resistance to both 1-β-D-arabinofuranosyladenine and 1-β-D-arabinofuranosylcytosine.These results indicate that an enzyme involved in production of dATP or its regulation is altered in these mutants. It is suggested that dATP competes with aphidocolin at its killing site or that dATP reverses the effect of aphidicolin by some unknown mechanism $\text{in}\text{vivo}$.     

Activation of Sepharose with epichlorohydrin and subsequent immobilization of ligand for affinity adsorbent.

The optimal conditions for the activation of Sepharose by epichlorohydrin and subsequent immobilization of ligands were investigated. Under the optimal conditions for activation, namely, 30% Sepharose-5% epichlorohydrin-0.4 M NaOH, 40 degrees C, 2 h, the maximum amount of epoxy group was introduced into Sepharose with low cross-linking. The absorbents obtained by using N-acetyl-D-glucosamine, tri-N-acetylchitotriose, and glycoprotein as a ligand exhibited no nonspecific adsorption and good permeability for the high molecular substance to be purified, and were stable in an alkaline solution. Solanum tuberosum agglutinin was specifically adsorbed on a tri-N-acetylchitotriose-Sepharose column and was quantitatively recovered by elution with 0.2 M ammonia solution. Furthermore, the column could be repeatedly used under these conditions without reduction of its capacity.     

The anticoagulant properties of mast cell product, chondroitin sulphate E

The anticoagulant potency in vitro of chondroitin sulphate E has been found to be similar to that of the heparinoids. In purified systems chondroitin sulphate E was shown to be principally an activator of heparin cofactor II. Maximum acceleration of heparin cofactor II:thrombin interaction was 185-fold (9.3 X 10(7) M-1 min-1), antithrombin III:thrombin interaction was 11-fold (4.16 X 10(6) M-1 min-1) and antithrombin III:factor Xa was 146-fold (3.86 X 10(6) M-1 min-1). Chondroitin sulphate E was observed to prolong the thrombin clotting time of fibrinogen in the absence of antithrombin III and heparin cofactor II. The effect appeared to be related to interference in thrombin:fibrinogen interaction rather than in fibrin monomer polymerization.     

Group X secretory phospholipase A2 can induce arachidonic acid release and eicosanoid production without activation of cytosolic phospholipase A2 alpha

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.     

Parkin stabilizes PINK1 through direct interaction

Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic; however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson’s disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD.     

Isolation of mouse FM3A cell mutants with thermolabile thymidylate synthetase by resistance to aphidicolin

Of 625 aphidicolin-resistant clones selected at 33.5°C from mutagenized mouse FM3A cells, 13 clones could not grow at 39.5°C. Five of these clones, chosen at random, resumed growth at 39.5°C when thymidine was added to the culture medium. In hybrids, conditional thymidine auxotrophy was a recessive trait, but aphidicolin-resistance was either a codominant or recessive one depending on the mutant clone used.Thymidylate synthetase activity in crude extracts of these mutants was completely inactivated by preincubation for 30 min at 42°C, whereas that of the parent cells was not affected by the same treatment. Thus, the temperature-sensitive growth of the mutants described here seems to be due to this heat-sensitive thymidylate synthetase.     

Nucleotide sequence of a functional cDNA for human thymidylate synthase.

We have determined the nucleotide sequence of a cDNA clone, pcHTS-1, encoding human thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) which was previously isolated from a human fibroblast expressible cDNA library and functional in mouse cells. The 1.6 kilobase cDNA insert of pcHTS-1 encodes a subunit protein of 313 amino acid (Mr = 35,706) and its predicted amino acid sequence is highly conserved in many regions including folylpolyglutamate and 5-fluoro-2'-deoxyuridylate binding sites, when compared with those of Lactobacillus casei, Escherichia coli, and bacteriophage T4. The cDNA contains in its 5'-untranslated region a triple tandemly repeated sequence consisting of 90 nucleotides, which starts immediately upstream of the ATG initiator codon, is very high in G+C content (80%), and can form three possible interconvertible stem-loop structures.