Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.     

A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family.

A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.     

The hypotrophic axonopathy mutant in Japanese quail.

A new behavioral mutant showing either head or body quivering, or both, was found in Japanese quail. This trait was characterized by neurofilament deficiency in the axons of the cervical spinal cord and the optic and sciatic nerves and was named "hypotrophic axonopathy." This character was shown to be controlled by an autosomal recessive gene, for which the gene symbol hax was proposed.     

Cell-division inhibitor produced by a killer strain of cellular slime mold Polysphondylium pallidum

The killer strain CK-8 of cellular slime mold Polysphondylium pallidum produces a cell-division inhibitor, in addition to a killer factor. This inhibitor represses cell division of many strains and species of cellular slime molds, except CK-8 itself and its complementary mating-type strain. It is sensitive to both heat and trypsin, and capable of binding to Con A. Its apparent molecular mass is more than 100 kDa. Repression of cell division by this inhibitor is reversed by trypsin treatment of the cells.     

RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.

An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

(-)-Epigallocatechin gallate amplifies interleukin-1-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells

We previously reported that interleukin-1 (IL-1), a potent bone resorptive cytokine, stimulates the synthesis of interleukin-6 (IL-6) via activation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that AMP-activated protein kinase (AMPK) negatively regulates the IL-1-induced IL-6 synthesis through the inhibitor of κB (IκB)/nuclear factor-κB (NF-κB) pathway. On the other hand, it is recognized that catechin possesses a beneficial property for bone metabolism. Among them, (-)-epigallocatechin gallate (EGCG) is an abundant and major bioactive component. In the present study, we investigated the effect of EGCG on the IL-1 stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. EGCG significantly enhanced the IL-1-stimulated IL-6 synthesis in a dose-dependent manner in the range between 50 and 100 μM. EGCG increased the mRNA levels of IL-6 stimulated by IL-1. IL-1-induced phosphorylation of IκB and NF-κB were suppressed by EGCG. On the other hand, EGCG failed to affect the IL-1-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and AMPK. These results strongly suggest that EGCG enhances IL-1-stimulated IL-6 synthesis through inhibiting the AMPK-IκB/NF-κB pathway at the point between AMPK and IκB/NF-κB in osteoblasts.     

Heme-binding properties of heme detoxification protein from Plasmodium falciparum

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.