Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans

We designed an automatic system to measure body length, diameters and volume of a C. elegans worm. By using this system, mutants with an increased body volume exceeding 50% were isolated. Four of them are grossly normal in morphology and development, grow longer to be almost twice as big, and have weak egg-laying defects and extended lifespan. All the four mutants have a mutation in the egl-4 gene. We show that the egl-4 gene encodes cGMP-dependent protein kinases. egl-4 promoter::gfp fusion genes are mainly expressed in head neurons, hypodermis, intestine and body wall muscles. Procedures to analyze morphology and volume of major organs were developed. The results indicate that volumes of intestine, hypodermis and muscle and cell volumes in intestine and muscle are increased in the egl-4 mutants, whereas cell numbers are not. Experiments on genetic interaction suggest that the cGMP-EGL-4 signaling pathway represses body size and lifespan through DBL-1/TGF-beta and insulin pathways, respectively.     

Direct interaction of the Golgi membrane with the endoplasmic reticulum membrane caused by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, blocks protein transport from the endoplasmic reticulum (ER) to the Golgi complex and induces the redistribution of Golgi proteins into the ER. We investigated characteristics of NDGA-induced retrograde movement of the Golgi proteins to the ER. At an early stage of incubation of cells with NDGA, the Golgi complex formed convoluted membrane aggregates. Electron microscopy revealed that these aggregates directly interact en bloc with the ER membrane. The direct interaction and subsequent incorporation of the Golgi proteins into the ER were found to be temperature-dependent. The protein of ER-Golgi intermediate compartment (ERGIC), ERGIC53, was rapidly accumulated in the Golgi upon treatment with NDGA. This accumulation was significantly inhibited by low temperature at 15 degrees C. Under the condition, the redistribution of the Golgi proteins into the ER as well as the direct interaction between the ER and the Golgi by NDGA were also inhibited, suggesting an important role of the ERGIC in the retrograde movement. In contrast, the low temperature did not inhibit formation of the Golgi aggregates by NDGA. Taken together, these results suggest that NDGA causes the redistribution of the Golgi proteins into the ER through the direct connections between the Golgi, the ERGIC, and the ER.     

A novel cyclic peptide immunization strategy for preventing HIV-1/AIDS infection and progression

A novel synthetic peptide immunogen targeting the human immunodeficiency virus type-1 (HIV-1) coreceptor CXCR4 was evaluated for its capacity to induce CXCR4-specific antibodies with anti-HIV-1 activity in BALB/c mice and cynomolgus monkeys. A cyclic closed-chain dodecapeptide mimicking the conformation-specific domain of CXCR4 (cDDX4) was prepared in which Gly-Asp, as the dipeptide forming a spacer arm, links the amino and carboxyl termini of the decapeptidyl linear chain (linear DDX4, Asn176 to Ile185) derived from the undecapeptidyl arch (UPA; Asn176 to Cys186) of extracellular loop 2 (ECL-2) in CXCR4. Immunization of BALB/c mice with cDDX4 conjugated with a multiple-antigen peptide (cDDX4-MAP) induced conformational epitope-specific antibodies, and monoclonal antibody IA2-F9 reacted with cDDX4, but not with linear DDX4, as determined by real-time biomolecular interaction analysis using surface plasmon resonance. The antibody also reacted with cells expressing CXCR4 but not with cells expressing the other HIV coreceptor, CCR5. Furthermore, the antibody inhibited the replication of HIV-1 X4 virus (using CXCR4), as shown by an infection assay using both MAGIC-5 cells and MT4 cells, but not that of HIV-1 R5 virus (using CCR5). The antibody weakly interfered with chemotaxis induced by stromal cell-derived factor-1 alpha in THP-1 cells or moderately inhibited the chemotaxis of Molt4#8 cells under the same conditions. In addition, immunization of cynomolgus monkeys also induced cDDX4-specific antibodies with anti-HIV activity. Taken together, these results indicate that cDDX4 conjugated with a multi-antigen peptide induces the conformational epitope-specific antibodies to the undecapeptidyl arch of CXCR4 may be a novel candidate immunogen for preventing disease progression in HIV-1-infected individuals.     

Role of the PLC-related, catalytically inactive protein p130 in GABA(A) receptor function

The protein p130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for gamma-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABA(A) receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABA(A) receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the gamma2 subunit of the GABA(A) receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl- current by Zn2+ or diazepam, both of which act at GABA(A) receptors containing gamma subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABA(A) receptors, especially in response to the agents acting on a gamma2 subunit.     

Novel strategy for anti-HIV-1 action: selective cytotoxic effect of N-myristoyltransferase inhibitor on HIV-1-infected cells

A 33-kDa protein component of the oxygen-evolving complex in photosystem II is essential for photosynthesis, and it has been believed that mutants with deletion of this 33-kDa protein are not found in higher plants. We report here the first isolation of an Arabidopsis thaliana mutant with a defect in one of the genes for the 33-kDa proteins, psbO, and an intact gene (psbO2). This mutant showed considerable growth retardation, suggesting that there is a functional difference between psbO and psbO2.     

Relationship Between 2,5-Hexanedione Concentrations in Nerve,Serum, and Urine Alone or Under Co-Treatment With Different Doses of Methyl Ethyl Ketone,Acetone, and Toluene

To ascertain the relationship among 2,5-hexanedione (2,5-HD) concentrations in nerve, serum and urine, rats were injected subcutaneously with 2.6 mmol/kg 2,5-HD alone, or together with 2.6 or 13.0 mmol/kg of methyl ethyl ketone, acetone and toluene. 2,5-HD concentrations in sciatic nerve (NC), serum (SC) and urine (UC) were determined, and the linear regression between each two of NC, SC, and UC were calculated. There was good correlation between NC and SC, SC and UC in the 2,5-HD alone group, and good correlation between NC and SC in the co-treated groups. Co-treatment solvent had little effect on the relationship between SC and NC. 13.0 mmol/kg co-treated solvent tended to decrease the regression coefficients compared with 2.6 mmol/kg co-treated solvent. These results show that SC can be used in estimating NC in the 2,5-HD alone or co-treated groups, and UC can be used in estimating SC in the 2,5-HD alone group.     

Membrane association of a new inositol 1,4,5-trisphosphate binding protein, p130 is not dependent on the pleckstrin homology domain.

The 130-kDa protein was isolated as a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding protein from rat brain and was molecularly cloned to be found similar to phospholipase C-delta 1 (Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M., 1992. Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol, J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M., 1996. A new inositol 1,4,5-trisphosphate binding protein similar to phospholipase C-delta 1, Biochem. J. 313, 319-325). The 130-kDa protein and its deleted protein expressed in COS-1 cells were seen in both the membrane and the cytosol fractions. Truncation of 232 residues from the N-terminus, the protein molecule lacking the pleckstrin homology (PH) domain was also localized in the membrane fraction as much as seen with a full-length protein and other deleted proteins, thereby indicating that the PH domain is not primarily involved in the membrane localization. The addition of Mg2+ to homogenates of COS-1 cells caused the translocation of expressed proteins from the cytosol to the membrane fraction, yet further addition of AlF4- which induced the activation of GTP binding proteins did not cause a further translocation. The protein translocated to the membrane by the addition of Mg2+ was hardly extracted with Triton X-100. The inclusion of Ins(1,4,5)P3 or phosphatidylinositol 4,5-bisphosphate in cell homogenates caused the very small reduction in the amounts of membrane-associated proteins expressed by some constructs. These results indicate that (i) the PH domain is not primarily involved in the membrane localization of the 130-kDa protein, (ii) the activation of GTP binding protein does not appear to cause the translocation of the 130-kDa protein, and (iii) intrinsic phosphatidylinositol 4,5-bisphosphate present in the membrane appears to be involved in the membrane association of the 130-kDa protein to a very small extent, probably through the binding site in the PH domain.     

Fasting Enhances TRAIL-Mediated Liver Natural Killer Cell Activity via HSP70 Upregulation

Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)⁺ and CD69⁺ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL⁻ natural killer cells that were adoptively transferred into Rag-2^−/− γ chain^−/− mice could convert into TRAIL⁺ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.     

Depletion of vesicle-tethering factor p115 causes mini-stacked Golgi fragments with delayed protein transport

Depletion of p115 with small interfering RNA caused fragmentation of the Golgi apparatus, resulting in dispersed distribution of stacked short cisternae and a vesicular structure (mini-stacked Golgi). The mini-stacked Golgi with cis- and trans-organization is functional in protein transport and glycosylation, although secretion is considerably retarded in p115 knockdown cells. The fragmented Golgi was further disrupted by treatment with breferdin A and reassembled into the mini-stacked Golgi by removal of the drug, as observed in control cells. In addition, p115 knockdown cells maintained retrograde transport from the Golgi to the endoplasmic reticulum, although the rate was not as efficient as in control cells. While no alternation of microtubule networks was found in p115 knockdown cells, the fragmented Golgi resembled those in cells treated with anti-microtubule drugs. The results suggest that p115 is involved in vesicular transport between endoplasmic reticulum and the Golgi, along with microtubule networks.     

Multiple isoforms of cyclophilin A associated with human immunodeficiency virus type 1

It is well-known that a peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA) is incorporated into Human immunodeficiency virus type 1 (HIV-1) particle. The proteome analysis of the purified HIV-1 strain LAV-1 (HIV-1(LAV-1)) reveals that three isoforms of CyPA with an isoelectric point (pI) of 6.00, 6.40, and 6.53 are inside the viral membrane and another isoform with a pI of 6.88 is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N-acetylated. The mechanisms that permit the redistribution of CyPA with a pI of 6.88 on the viral surface have not yet been clarified, but it penetrates the viral membrane after budding.