Double dioxygenation by mouse 8S-lipoxygenase: specific formation of a potent peroxisome proliferator-activated receptor alpha agonist

Mouse 8S-lipoxygenase (8-LOX) metabolizes arachidonic acid (AA) specifically to 8S-hydroperoxyeicosatetraenoic acid (8S-HPETE), which will be readily reduced under physiological circumstances to 8S-hydroxyeicosatetraenoic acid (8S-HETE), a natural agonist of peroxisome proliferator-activated receptor alpha (PPAR alpha). Here, we investigated whether 8-LOX could further oxygenate AA and whether the products could activate PPARs. The purified recombinant 8-LOX converted AA exclusively to 8S-HPETE and then to (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicosatetraenoic acid (8S,15S-diHPETE). The kcat/Km values for 8S-HPETE and AA were 3.3x10(3) and 2.7x10(4) M(-1) s(-1), respectively. 8-LOX also dioxygenated 8S-HETE and 15S-H(P)ETE specifically to the corresponding 8S,15S-disubstituted derivatives. By contrast, 15-LOX-2, a human homologue of 8-LOX, produced 8S,15S-diH(P)ETE from 8S-H(P)ETE but not from AA nor 15S-H(P)ETE. 8S,15S-diHETE activated PPAR alpha more strongly than 8S-HETE did. The present results suggest that 8S,15S-diH(P)ETE as well as 8S-H(P)ETE would contribute to the physiological function of 8-LOX and also that 8-LOX can function as a potential 15-LOX.     

Synergistic effect of interleukin-15 and interleukin-12 on antitumor activity in a murine malignant pleurisy model

 Interleukin(IL)-15, which uses IL-2 receptor (R) β and γ chains for signal transduction, shares many of the biological activities of IL-2. We examined the effects of exogenous IL-15 on protection in a murine malignant pleurisy model using BALB/c mice and syngeneic MethA fibrosarcoma (MethA). Intrapleural administration of IL-15 significantly prolonged the survival time of mice after an intrapleural inoculation of MethA, whereas the same dose of IL-2 did not. The in vivo antitumor effect of IL-15 was synergistically enhanced by additive administration of IL-12. Combination therapy of IL-15 and IL-12 protected mice from death from bloody pleural fluid. Such treatment induced marked increases in the number of CD3^-IL-2Rβ⁺ cells corresponding to natural killer (NK) cells and the production of interferon γ (IFNγ) by T cells in the thoracic exudate cells (TEC). Administration of anti-IFNγ mAb partly inhibited the protective effect of a combination of IL-15 and IL-12. A tumor-neutralizing (Winn) assay revealed that the antitumor activity of effector cells in the TEC was abrogated by treatment with anti-CD8 mAb or anti-asialoGM1 Ab plus complement. Thus, treatment with IL-15 in combination with IL-12 may enhance the activities of NK and CD8⁺ T cells in the TEC, providing strong antitumor activity against the malignant pleurisy. These results suggest that IL-15 together with IL-12 may have potential for the immunotherapy of some types of malignant pleurisy. Received: 13 July 1999 / Accepted: 3 December 1999     

Evolution of Serpin Specificity: Cooperative Interactions in the Reactive-Site Loop Sequence of Antithrombin Specifically Restrict the Inhibition of Activated Protein C

Protease cascades and their inhibitors are a common feature of many biological regulatory systems, and the various components of such cascades have been subjected to a long and concerted evolution. We present here evidence that in the coagulation cascade, the sequence of the protease-binding reactive-site loop of antithrombin has evolved such that the majority of its residues has been acquired not for the efficient inhibition of its target proteases, thrombin and factor Xa, but to avoid the inhibition of activated protein C (APC). We substituted residues of the reactive-site loop of antithrombin into α₁-antitrypsin and tested the chimeras against thrombin, factor Xa, and APC. With respect to factor Xa and thrombin, the difference in association rate between the fastest and the slowest inhibitors was 5.5- and 88-fold, respectively. However, with respect to APC the difference was 12,500-fold. While most of the variation in the inhibition rates of thrombin could be accounted for by P2 Gly-to-Pro substitutions, for APC almost every residue had an effect on inhibition. In 22 of 25 direct comparisons of antitrypsin residues with antithrombin residues, either singly or in blocs, the antithrombin residues caused a decrease in the rate of inhibition of APC. The antithrombin residue Asn393, at position P′3, emerged as particularly important for avoiding the inhibition of APC, however, its 190-fold effect was seen only when in conjunction with antithrombin P7 to P′2 residues. Cooperative effects among residues of the reactive-site loop thus emerged as critical for restricting the activity of this sequence against APC. Received: 15 November 1999 / Accepted: 2 August 2000     

Expression of testicular fatty acid-binding protein PERF 15 during germ cell apoptosis

PERF 15 is a testicular germ cell specific fatty acid-binding protein (FABP) isolated from rat. Indirect immunofluorescent analysis of juvenile rat testis showed that there were some strongly PERF 15-positive spermatocytes. These cells showed unclear nuclear structure and were predicted to undergo apoptosis. Apoptosis in germ cells is an important regulatory event to limit the number of germ cells in the seminiferous epithelium, but the physiological significance and molecular mechanisms of this testicular germ cell apoptosis are poorly understood. To determine whether PERF 15 participates in germ cell apoptosis, juvenile rat testis was examined by immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) methods. Strongly PERF 15-positive cells and TUNEL-positive cells were co-localized in adjacent sections. Exposure to methoxyacetic acid (MAA), known to induce apoptosis in spermatocytes, increased the number of strongly PERF 15-positive cells in 25-day-old rats' testes. Therefore, it seems that PERF 15 is involved in both spermatogenesis and testicular germ cell apoptosis.     

Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization

Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.     

Natural abundance of 15N in actinorhizal plants and nodules

Substantial enrichment of some plant parts in ¹⁵N relative to the rest of the plant is unusual, but is found in the nitrogen-fixing nodules of many legumes. A range of actinorhizal plants was surveyed to determine whether the nodules of any of them are also substantially enriched in ¹⁵N. The nonlegume Parasponia, nodulated by a rhizobium, was also included. Four of the actinorhizal genera and Parasponia were grown in N-free culture, and three actinorhizal genera were collected from the field. Nodules of Parasponia, Casuarina and Alnus were¹⁵N enriched relative to other plant parts, but only Parasponia approached the degree of enrichment found in some legume nodules. The nodules of Datisca, Myrica, Elaeagnus, Shepherdia, and Coriaria were depleted in ¹⁵N. Thus many actinorhizal nodules are depleted in ¹⁵N compared to other plant parts and enrichment is modest when it does occur. Whole plant ¹⁵N content (¹⁵N) in four actinorhizal plants and Parasponia showed a relatively narrow range of –1.41 to –1.90. Hence regardless of the degree of nodule enrichment or depletion, whole plant ¹⁵N content appears to vary little in plants grown in N-free culture.     

Dynamics of the Hck-SH3 domain: comparison of experiment with multiple molecular dynamics simulations

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481-493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 5N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well-defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5-10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.