Clinical Significance of sIL-2R Levels in B-Cell Lymphomas

Elevated soluble interleukin-2 receptor (sIL-2R) in sera is observed in patients with malignant lymphoma (ML). Therefore, sIL-2R is commonly used as a diagnostic and prognostic marker for ML, but the mechanisms responsible for the increase in sIL-2R levels in patients with B-cell lymphomas have not yet been elucidated. We first hypothesized that lymphoma cells expressing IL-2R and some proteinases such as matrix metalloproteinases (MMPs) in the tumor microenvironment can give rise to increased sIL-2R in sera. However, flow cytometric studies revealed that few lymphoma cells expressed IL-2R α chain (CD25) in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and most CD25-expressing cells in the tumor were T-cells. Distinct correlations between CD25 expression on B-lymphoma cells and sIL-2R levels were not observed. We then confirmed that MMP-9 plays an important role in producing sIL-2R in functional studies. Immunohistochemical (IHC) analysis also revealed that MMP-9 is mainly derived from tumor-associated macrophages (TAMs). We therefore evaluated the number of CD68 and CD163 positive macrophages in the tumor microenvironment using IHC analysis. A positive correlation between the levels of sIL-2R in sera and the numbers of CD68 positive macrophages in the tumor microenvironment was confirmed in FL and extranodal DLBCL. These results may be useful in understanding the pathophysiology of B-cell lymphomas.     

Sip1, an AP-1 Accessory Protein in Fission Yeast,Is Required for Localization of Rho3 GTPase

Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3 ⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.     

Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1−/− mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.     

3'-Azido-2',3'-dideoxynucleoside 5'-triphosphates inhibit telomerase activity in vitro, and the corresponding nucleosides cause telomere shortening in human HL60 cells

Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3′-Azido-3′-deoxythymidine 5′-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3′-down azido group: 3′-azido-2′,3′-dideoxyguanosine (AZddG) 5′-triphosphate (AZddGTP), 3′-azido-2′,3′-dideoxy-6-thioguanosine (AZddSG) 5′-triphosphate (AZddSGTP), 3′-azido-2′,3′-dideoxyadenosine (AZddA) 5′-triphosphate (AZddATP) and 3′-azido-2′,3′-dideoxy-2-aminoadenosine (AZddAA) 5′-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3′-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases α and δ, suggesting that AZddGTP is a selective inhibitor of telomerase.

We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.

     

Micromechanics of the Vertebrate Meiotic Spindle Examined by Stretching along the Pole-to-Pole Axis

The meiotic spindle is a bipolar molecular machine that is designed to segregate duplicated chromosomes toward the opposite poles of the cell. The size and shape of the spindle are considered to be maintained by a balance of forces produced by molecular motors and microtubule assembly dynamics. Several studies have probed how mechanical perturbations of the force balance affect the spindle structure. However, the spindle’s response to a stretching force acting at the spindle pole and along its long axis, i.e., the direction in which chromosomes are segregated, has not been examined. Here, we describe a method to apply a stretching force to the metaphase spindle assembled in Xenopus egg extracts and measure the relationship between the force and the three-dimensional deformation of the spindle. We found that the spindle behaves as a Zener-type viscoelastic body when forces are applied at the spindle pole, generating a restoring force for several minutes. In addition, both the volume of the spindle and the tubulin density are conserved under the stretching force. These results provide insight into how the spindle size is maintained at metaphase.     

Measurement of Dipole Potential in Bilayer Lipid Membranes by Dielectric Spectroscopy

Planar bilayer lipid membranes formed from egg phosphatidylcholine in aqueous media containing the lipophilic anion, dipicrylamine (DPA), were studied by dielectric spectroscopy over a frequency range of 10 Hz–10 MHz. The membranes showed dielectric relaxation due to the translocation of DPA between the membrane interfaces. Incorporating either cholesterol or 6-ketocholestanol into the membranes increased the characteristic frequency of the relaxation, which is proportional to the translocation rate constant of DPA. The results suggested that the sterol dipoles induced positive potential changes within the membrane interior. The changes of the dipole potential were 70 mV for cholesterol and 150 mV for 6-ketocholestanol when the sterol mole fraction was 0.67. The opposite effect was caused by phloretin added to the aqueous media, and the maximum dipole potential change was ?90 mV at 100 μM.     

Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice

Aims

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a^−/−) mice and human Akrla-transgenic (Akr1a^tg/+) mice experience different anesthetic actions from pentobarbital—prolonged in Akr1a-knockout (Akr1a^−/−) mice and shortened in human Akrla-transgenic (Akr1a^tg/+) mice.

Main methods

We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice.

Key findings

Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a^−/− mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a^−/− mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics.

Significance

Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients.     

Trichinella nativa and Trichinella T9 in the Hokkaido island, Japan

Trichinella sp. muscle larvae were isolated from the thigh muscle of two red foxes (Vulpes vulpes) captured in Sapporo and Otofuke, Hokkaido, Japan, in 2003. Multiplex PCR designed for genotyping the genus Trichinella revealed that the Sapporo isolate showed a specific pattern to T. britovi complex (T. britovi, Trichinella T8 and Trichinella T9) and the Otofuke isolate showed that to T. nativa. Nucleotide sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) of the Sapporo isolate showed the highest similarity to those of Trichinella T9, a species detected in the mainland of Japan. This study shows that both T. nativa and Trichinella T9 are circulating in wildlife of the Hokkaido island.