Purified group X secretory phospholipase A(2) induced prominent release of arachidonic acid from human myeloid leukemia cells

Group X secretory phospholipase A(2) (sPLA(2)-X) possesses several structural features characteristic of both group IB and IIA sPLA(2)s (sPLA(2)-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA(2)-X, the preparation of its antibody, and the purification of native sPLA(2)-X. The affinity-purified sPLA(2)-X protein migrated as various molecular species of 13-18 kDa on SDS-polyacrylamide gels, and N-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH(2)-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA(2)-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH(2) termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA(2)-X has a relatively weak potency compared with the mature protein. The mature sPLA(2)-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA(2) groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA(2)-IB and -IIA with concomitant production of prostaglandin E(2). A prominent release of arachidonic acid was also observed in sPLA(2)-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA(2)-X is a unique N-glycosylated sPLA(2) that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA(2)-IB and -IIA.     

Crystallographic studies on damaged DNAs. II. N(6)-methoxyadenine can present two alternate faces for Watson-Crick base-pairing, leading to pyrimidine transition mutagenesis

In a previous paper, 2'-deoxy-N(6)-methoxyadenosine (mo(6)A) was shown to form a mismatch base-pair with 2'-deoxycytidine with a Watson-Crick-type geometry. To fully understand the structural basis of genetic mutations with damaged DNA, it is necessary to examine whether the methoxylated adenine residue still has the ability to form the regular Watson-Crick pairing with a thymine residue. Therefore, a DNA dodecamer with the sequence d(CGCGmo(6)AATTCGCG) has been synthesized and its crystal structure determined. The methoxylation has no significant effect on the overall DNA conformation, which is that of a standard B-form duplex. The methoxylated adenine moieties adopt the amino tautomer with an anti conformation around the C(6)-N(6) bond to the N(1) atom, and they form a Watson-Crick base-pair with thymine residues on the opposite strand, similar to an unmodified adenine residue. It is concluded that methoxylated adenine can present two alternate faces for base-pairing, thanks to the amino<-->imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.     

Transient hyper-17-OHPnemia unrelated to cross-reactions with residual fetal adrenal cortex products

OBJECTIVE: To clarify the pathogenesis of transient hyper-17alpha-hydroxyprogesteronemia, we initiated a laboratory investigation in a pre-term infant with persistently high serum 17alpha-hydroxyprogesterone (17-OHP) until 2 months of age. METHODS: Serum 17-OHP level was measured by high-performance liquid chromatography and radioimmunoassay, and gene analysis of CYP21A2 (21-hydroxylase) was performed. RESULT: Serum 17-OHP level on the 29th day of life was 25.4 ng/ml, and the urinary steroid profile showed low pregnanetriolone. Gene analysis of 21-hydroxylase disclosed no mutation, and 17-OHP normalized by 3 months of age without specific treatment. CONCLUSION: Transient elevations in 17-OHP, which do not appear related to cross-reactions with products of a residual fetal adrenal cortex, may occur in the first few months of life.     

Over-expression of the testis-specific gene TSGA10 in cancers and its immunogenicity

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real-time RT-PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over-expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.     

Attenuation of a delayed increase in the extracellular glutamate level in the peri-infarct area following focal cerebral ischemia by a novel agent ONO-2506

A novel agent, ONO-2506 [(R)-(-)-2-propyloctanoic acid, ONO Pharmaceutical Co. Ltd.] was previously shown to mitigate delayed infarct expansion through inhibition of the enhanced production of S-100beta, while inducing a prompt symptomatic improvement that attained a significant level as early as 24h after drug administration. To elucidate the mechanism underlying the prompt symptomatic improvement, the present study aimed to examine whether ONO-2506 modulates the level of extracellular glutamate ([Glu]e) in the rat subjected to transient middle cerebral artery occlusion (tMCAO). In this model, it had been shown that ONO-2506 reduces the infarct volume, improves the neurological deficits, and enhances the mRNA expression of glial glutamate transporters (GLT-1 and GLAST). The [Glu]e levels in the ischemic cortices were continuously measured using intracerebral microdialysis. The alterations in the [Glu]e levels in the sham-operated and tMCAO-operated groups with or without drug administration were compared. In the tMCAO groups, the [Glu]e level increased during tMCAO to a similar extent, returned to normal on reperfusion, and increased again around 5h. In the saline-treated group, however, the [Glu]e level further increased from 15 h on to reach about 280% of the normal level at 24h. This secondary increase in the [Glu]e level in the late phase of reperfusion was prevented by ONO-2506. The intracerebral infusion of glutamate transporter inhibitor, l-trans-pyrrolidine-2,4-dicarboxylic acid, at 24h after tMCAO induced an increase in the [Glu]e level, which was marked in both the sham-operated and ONO-2506-treated groups, but much less pronounced in the saline-treated group. The above results suggest that functional modulation of activated astrocytes by pharmacological agents like ONO-2506 may inhibit the secondary rise of [Glu]e level in the late phase of reperfusion, leading to amelioration of delayed infarct expansion and neurological deficits.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals. Part 5

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

TetraThymosinbeta is required for actin dynamics in Caenorhabditis elegans and acts via functionally different actin-binding repeats

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinbeta expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinbeta has four repeats, each related to the actin monomer-sequestering protein thymosinbeta 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinbeta binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinbeta may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinbeta is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinbeta is of crucial importance at specific developmental stages requiring actin polymerization.