Utilization of the Porcine System to Study Lymphotoxin-β Regulation in Intestinal Lymphoid Tissue

Lymphotoxin-β (LT-β) has been suggested to be a regulator of secondary lymphoid structure development. In the present study, we isolated porcine LT-β (poLT-β) from adult swine spleens. The open reading frame encoded a predicted 246-amino acid polypeptide exhibiting higher similarity to the human than the mouse LT-β protein. Expression of LT-β mRNA in various swine tissues was analyzed by real-time PCR, and it was found to be higher in the ileal Peyer's patches (Pps) of adults than in newborns. In addition, ligand stimulation of toll-like receptors 2, 4, and 9, which are activated by bacterial components, increased LT-β expression only in neonatal ileal Pps. These results suggest that colonization by commensal bacteria may affect the maturation of neonatal ileal Pps by the induction of LT-β via toll-like receptors. LT-β may therefore be useful for studying the development of the intestinal immune system at parturition in both swine and humans.     

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Store-operated Ca²⁺ entry (SOCE) is a universal mechanism to increase intracellular Ca²⁺ concentrations in non-excitable cells. It is initiated by the depletion of ER Ca²⁺ stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca²⁺ release activated Ca²⁺ (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca²⁺ influx and CRAC currents (I_CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I_CRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca²⁺ stores.     

A murine model of neonatal diabetes mellitus in Glis3-deficient mice

Glis3 is a member of the Gli-similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3-deficient mice by gene-targeting. The Glis3^−/− mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulin-producing cell formation. The pancreatic phenotypes indicate that the Glis3-deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.     

Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage

RSΒ1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The RSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of RSB1 were demonstrated.     

Establishment and characterization of the Bombyx mandarina cell line

A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2 years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 °C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei’s genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.     

Thermal Response with Exothermic Effects of β2-Microglobulin Amyloid Fibrils and Fibrillation

Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of β₂-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid β-peptides but also during the processes of the temperature-induced conversion of β₂-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules.     

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.HIV-1³ is an enveloped retrovirus, which acquires its lipid envelope by budding from the plasma membrane of the infected host cell. Several reports have shown that the viral membrane is enriched in sphingomyelin (SM), including the unusual sphingolipid dihydrosphingomyelin (DHSM) and collectively referred to as sphingomyelins (SMs), glycosphingolipids, cholesterol (CHOL), saturated phosphoglycerolipids and phosphoinositides (1–4). Moreover the CHOL/phospholipid and protein/lipid ratios of the HIV-1 membrane are high, corresponding to a highly ordered membrane, and are presumed to be different from the overall host cell plasma membrane. Accordingly, the HIV-1 envelope has been considered to be a large raft-like membrane microdomain (3). This is in line with previous reports describing enrichment of raft markers in the HIV-1 membrane and its sensitivity to CHOL-depleting agents (5–9). Furthermore, HIV-1 glycoproteins have been suggested to localize within membrane rafts due to palmitoylation of two cysteines (10), and the main structural Gag protein has been shown to rapidly relocalize to detergent-resistant membranes after initial membrane binding (6).Membrane microdomains are dynamic assemblies resulting from the lateral interaction of lipids and proteins. Two phases coexist in the plasma membrane: the liquid-ordered phase (l_o), mainly composed of CHOL and sphingolipids (SPLs), and the liquid disordered phase (l_d), mainly composed of glycerophospholipids (11–13). In the activated state, l_o microdomains can coalesce and serve as platforms for membrane trafficking, signaling, and virus budding (14, 15). The first method to biochemically enrich membrane rafts was the purification of detergent-resistant membranes, based on their resistance to extraction with non-ionic detergent at 4 °C (16). However, this and other methods based on antibody or cholera toxin binding may lead to artificial aggregation of membrane microdomains and thus do not necessarily represent their native state (17, 18). For these reasons and because the association and dissociation of membrane microdomains appears to occur on a rapid time-scale and the raft size is too small to be optically resolved, the raft concept remains controversial. However, the determination of the HIV-1 lipidome, a native membrane purified without any detergent, has provided strong evidence for the existence of these microdomains (3).Fluorescent lipid analogs that partition preferentially into a specialized lipid phase could be an attractive tool to study membrane microdomains. However, partitioning of such dyes mainly depends on the local chemical environment and not on the phase state of the membrane (19–21). In contrast, Laurdan (6-dodecanoyl-2-dimethylaminonapthalene) is a lipophilic dye that binds to membranes independent of their phase state but reports the phase state by a change in its fluorescence emission (20). Laurdan exhibits a blue shift in its emission spectrum with increasing membrane condensation. This is caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. Thus, excitation of membrane bound Laurdan leads to two emission maxima representing differences in membrane lateral structure. Quantification of membrane order is achieved by computing the Generalized Polarization (GP) value, which is defined as normalized intensity ratio of the two emission channels. GP values range from +1 (most condensed) to −1 (most fluid). They are not biased by probe concentration, membrane ruffles, and surface modifications, such as lipoprotein binding. Furthermore, there is no preferential interaction with a specific lipid, fatty acid, or head group (20, 21). GP value correspondence to different lipid phases was estimated using liposomes with a composition similar to that of cellular membranes (22, 23). Using an equimolar mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine, CHOL, and SM as an l_o membrane, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as an l_d and solid ordered (s_o) phase, GP values below +0.25 were shown to correspond to the l_d phase, GP values between +0.25 and +0.5 to the l_o phase, and GP values above +0.5 to the s_o phase (22, 23).Laurdan has been extensively used to characterize domain formation and lateral lipid segregation in model membranes composed of different phospholipid mixtures or lipids extracted from cellular membranes (19, 22–25). It has also been used to study the membrane structure in living cells. Gaus and coworkers observed l_o domains enriched on membrane protrusions (filopodia), adhesion points, and cell-cell contacts (26). They also used Laurdan to address the physical properties of the plasma membrane around the T-cell receptor in activated T cells, observing larger and more stably ordered membrane domains at sites of T-cell activation (27). Quantitative determination of cellular plasma membrane order by fluorescence spectroscopy is complicated due to the rapid internalization and redistribution of the probes to other cellular membranes, making it difficult to interpret the fluorescence measurements over the whole cell. This problem is not encountered in purified virus particles, because they contain only a single membrane. We therefore developed an assay to study viral membrane lateral structure by fluorescence spectroscopy. For this purpose, isogenic HIV-1 particles were produced in two different cell lines, and their GP profiles were determined. In parallel, the lipid constituents were quantified by mass spectrometry. The viral membrane displayed a l_o structure in both cases, but this was more prominent for the virus derived from the T-cell line MT-4 compared with virus derived from 293T cells. The validity of this result was supported by comparing the lipidome of the two viruses, which revealed a significantly higher SMs/phosphatidylcholine (PC) ratio for the MT-4-derived virus. Accordingly, treatment with SM-binding toxins inactivated MT-4-derived virus more efficiently than 293T-derived virus, whereas both viruses exhibited similar infectivities before treatment. The reported approach allows rapid determination of differences in viral membrane order, permitting screening for compounds that perturb l_o domains, which may act as antivirals of a novel type.     

Activation of AMP-activated Protein Kinase Suppresses Oxidized Low-density Lipoprotein-induced Macrophage Proliferation

Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [³H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKα1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27^kip suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21^cip and p27^kip expression via AMPK activation, and small interfering RNA (siRNA) of p21^cip and p27^kip restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.     

Growth inhibitory activity of ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranosyl ester and ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidase

Exogenously applied ABA-β-d-glucopyranosyl ester (ABA-GE) inhibited shoot growth of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), Digitaria sanguinalis L., timothy (Pheleum pratense L.) and ryegrass (Lolium multiflorum Lam.) seedlings at concentrations greater than 0.1 μM. The growth inhibitory activity of ABA-GE on these shoots was 26–40% of that of (+)-ABA. ABA-β-d-glucosidase activities in these seedlings were 11–31 nmol mg⁻¹ protein min⁻¹. These results suggests that exogenously applied ABA-GE may be absorbed by plant roots and hydrolyzed by ABA-β-d-glucosidase, and liberated free ABA may induce the growth inhibition in these plants. Thus, although ABA-GE had been thought to be physiologically inactive ABA conjugate, ABA-GE may have important physiological functions rather than an inactive conjugated ABA form.     

Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.