Evidence for lysosomal processing of amyloid β-protein precursor in cultured cells

Amyloid -protein precursor (ABPP) of Alzheimer's disease (AD) represents a family of proteins which includes the parent protein which generates a small (4 kD) fragment that self-assembles to form amyloid fibrils in AD. Thus, the normal and abnormal proteolysis of ABPP may be directly relevant to AD pathogenesis. We have examined the accumulation of ABPP in cultured rodent and human neuronal cell lines in the presence and absence of a battery of protease inhibitors using immunohistochemistry and Western blot analysis. Here we present evidence for a lysosomal pathway for the turnover of ABPP and discuss the relevance of these results to plaque pathology and abnormal ABPP immunostaining in AD.Special issue dedicated to Dr. Paola S. Timiras     

Structures and synthesis of seed-germination inhibitors from hibiscus rosa-sinensis

8-Nonynoic, 9-decynoic acids and their methyl esters were isolated for the first time as natural products from Hibiscus rosa-sinensis. They were inhibitors of the germination of lettuce seeds and were synthesized via o-nitrophenyl selenides.     

Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.     

A phenol alloside from viburnum wrightii

A new phenol alloside, p-hydroxyphenyl β-D-alloside, has been isolated from the leaves of Viburnum wrightii in addition to several known compounds. The structures were elucidated by spectroscopic and chemical methods.     

Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart

Naked plasmid DNA (pDNA) and short interfering RNA (siRNA) duplexes were transduced into adult murine heart by means of sonoporation using the third-generation microbubble, BR14. Plasmid DNAs carrying luciferase, beta-galactosidase (beta-gal), or enhanced green fluorescent protein (EGFP) reporter genes were mixed with BR14 and injected percutaneously into the left ventricular (LV) cavity of C57BL/6 mice while exposed to transthoracic ultrasound at 1MHz for 60s. Sonoporation at an output intensity of 2.0W/cm(2) and a 50% pulse duty ratio resulted in the highest luciferase expression in the heart. Histological examinations revealed significant expression of the beta-gal and EGFP reporters in the subendocardial myocardium of LV. Intraventricular co-injection of siRNA-GFP and BR14 with concomitant ultrasonic exposure resulted in substantial reduction in EGFP expression in the coronary artery in EGFP transgenic mice. The present method may be applicable to gain-of-function and loss-of-function genetic engineering in vivo of adult murine heart.     

Folding transition into a loosely collapsed state in plasmid DNA as revealed by single-molecule observation

The conformational transition of a plasmid DNA, pGEG.GL3 (12.5 kbp, circular), induced by spermine(4+) was studied through the observation of individual DNA by fluorescence microscopy. We deduced the change in the hydrodynamic radius R(H) from an analysis of the Brownian motion of single DNA molecules. R(H) decreases in a continuous manner with an increase in spermine(4+), in contrast to the large discrete on/off change for long linear DNA. Just after the transition to the collapsed state, a small number of DNA molecules tend to form an assembly, which disperses in the bulk solution without precipitation.     

Initation of DNA replication in Escherichia coli. I. Characteristics of the initation process in dna mutants

Summary When a culture of E. coli strain carrying a temperature-sensitive DNA initiation mutation, dna-167 or dnaC2, is exposed to a nonpermissive temperature for a certain period of time, and then transferred back to a permissive temperature, DNA synthesis is resumed even in the presence of chloramphenicol. This shows that thermolabile components coded by either of these mutated genes can be reactivated after return to permissive temperatures, and consequently initiation of a new replication cycle can occur in the absence of concomitant protein synthesis in both strains. The reinitiation of replication occurring after lowering the temperature is sensitive to rifampicin in the dna-167 cells, but not in the dnaC2 mutant. The capacity for initiating a new round of replication is very labile in the dna-167 mutant, but not in the dnaC2 mutant, when a culture of the mutant is maintained at a nonpermissive temperature in the presence of rifampicin. Mechanisms of blocking of the initiation process with these mutants are discussed.After a prolonged exposure of an early-exponential phase culture to high temperatures, reinitiation of DNA replication never exceeds a doubling in both strains, when the temperature is lowered in the presence of chloramphenicol. However, after an exposure of a late-exponential phase culture to a nonpermissive temperature, more than one round of replication occurs in both strains even in the presence of chloramphenicol.     

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.     

Azedarachol,a steroid ester antifeedant from Melia azedarach var. japonica

A new steroid ester, azedarachol, from the root bark of Melia azedarach has been identified as an antifeedant against a Japanese insect pest and the structure has been assigned as 2α,3α,16β-trihydroxy-5α-pregnane 20R-methacrylate.     

Stimulation of sialidase activity during cell differentiation of human promyelocytic leukemia cell line HL-60

Sialidase activity of human promyelocytic leukemia cell line HL-60 was assayed by a modification of the fluorometric method using 4MU-NANA as a substrate. The pH optimum was 4.1 and the apparent Km value was 0.10 mM. When the cells were induced to differentiate into granulocytes by either retinoic acid or DMSO, sialidase activity increased markedly. After incubation of HL-60 cells with 1 μM retinoic acid for 6 days and with 1.3% DMSO for 8 days, 91% and 75% of total cells, respectively, differentiated into morphologically mature myeloid cells and the sialidase activity increased to 2.5–2.7 times as much as that of the corresponding controls. In other human myeloid leukemia cell lines, K562 and KG-1, the sialidase activity was found to be 1.5- and 3.8-fold that of HL-60, respectively.