Double-stranded RNA Mediates Selective Gene Silencing of Protein Phosphatase Type 1 Delta Isoform in HEK-293 Cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1δ) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP18. This RNA interference did not affect the expression of α and γ1 isoforms of PP1. Transfection of antisense RNA specific for PP1δ also suppressed the expression of PP1δ. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

Successful adjuvant bi-weekly gemcitabine chemotherapy for pancreatic cancer without impairing patients’ quality of life

Background

Although adjuvant gemcitabine (GEM) chemotherapy for pancreatic cancer is standard, the quality of life (QOL) in those patients is still impaired by the standard regimen of GEM. Therefore, we studied whether mild dose-intensity adjuvant chemotherapy with bi-weekly GEM administration could provide a survival benefit with acceptable QOL to the patients with pancreatic cancer.

Methods

After a phase I trial, an adjuvant bi-weekly 1,000 mg/m² of GEM chemotherapy was performed in 58 patients with pancreatic cancer for at least 12 courses (Group A). In contrast, 36 patients who declined the adjuvant bi-weekly GEM chemotherapy underwent traditional adjuvant 5FU-based chemotherapy (Group B). Careful periodical follow-ups for side effects of GEM and disease recurrence, and assessment of patients’ QOL using the EORTC QOL questionnaire (QLQ-C30) and pancreatic cancer-specific supplemental module (QLQ-PAN26) were performed. Retrospectively, the degree of side effects, patients’ QOL, compliance rate, disease-free survival (DFS), and overall survival (OS) in Group A were compared with those in Group B.

Results

No severe side effects (higher than Grade 2 according to the common toxicity criteria of ECOG) were observed, except for patients in Group B, who were switched to the standard GEM chemotherapy. Patients’ QOL was better in Group A than B (fatigue: 48.9 ± 32.1 versus 68.1 ± 36.3, nausea and vomiting: 26.8 ± 20.4 versus 53.7 ± 32.6, diarrhea: 21.0 ± 22.6 versus 53.9 ± 38.5, difficulty gaining weight: 49.5 ± 34.4 versus 67.7 ± 40.5, P < 0.05). Compliance rates in Groups A and B were 93% and 47%. There was a significant difference in the median DFS between both groups (Group A : B =12.5 : 6.6 months, P < 0.001). The median OS of Group A was prolonged markedly compared with Group B (20.2 versus 11.9 months, P < 0.005). For OS between both groups, univariate analysis revealed no statistical difference in 69-year-old or under females, and T1–2 factors, moreover, multivariate analysis indicated three factors, such as bi-weekly adjuvant GEM chemotherapy, T2 or less, and R0.

Conclusions

Adjuvant chemotherapy with bi-weekly GEM offered not only the advantage of survival benefits but the excellent compliance with acceptable QOL for postoperative pancreatic cancer patients.     

Substrate specificity,plasma membrane localization,and lipid modification of the aldehyde dehydrogenase ALDH3B1

The accumulation of reactive aldehydes is implicated in the development of several disorders. Aldehyde dehydrogenases (ALDHs) detoxify aldehydes by oxidizing them to the corresponding carboxylic acids. Among the 19 human ALDHs, ALDH3A2 is the only known ALDH that catalyzes the oxidation of long-chain fatty aldehydes including C16 aldehydes (hexadecanal and trans-2-hexadecenal) generated through sphingolipid metabolism. In the present study, we have identified that ALDH3B1 is also active in vitro toward C16 aldehydes and demonstrated that overexpression of ALDH3B1 restores the sphingolipid metabolism in the ALDH3A2-deficient cells. In addition, we have determined that ALDH3B1 is localized in the plasma membrane through its C-terminal dual lipidation (palmitoylation and prenylation) and shown that the prenylation is required particularly for the activity toward hexadecanal. Since knockdown of ALDH3B1 does not cause further impairment of the sphingolipid metabolism in the ALDH3A2-deficient cells, the likely physiological function of ALDH3B1 is to oxidize lipid-derived aldehydes generated in the plasma membrane and not to be involved in the sphingolipid metabolism in the endoplasmic reticulum.     

A Decreased Level of Serum Soluble Klotho Is an Independent Biomarker Associated with Arterial Stiffness in Patients with Chronic Kidney Disease

Background

Klotho was originally identified in a mutant mouse strain unable to express the gene that consequently showed shortened life spans. In humans, low serum Klotho levels are related to the prevalence of cardiovascular diseases in community-dwelling adults. However, it is unclear whether the serum Klotho levels are associated with signs of vascular dysfunction such as arterial stiffness, a major determinant of prognosis, in human subjects with chronic kidney disease (CKD).

Methods

We determined the levels of serum soluble Klotho in 114 patients with CKD using ELISA and investigated the relationship between the level of Klotho and markers of CKD-mineral and bone disorder (CKD-MBD) and various types of vascular dysfunction, including flow-mediated dilatation, a marker of endothelial dysfunction, ankle-brachial pulse wave velocity (baPWV), a marker of arterial stiffness, intima-media thickness (IMT), a marker of atherosclerosis, and the aortic calcification index (ACI), a marker of vascular calcification.

Results

The serum Klotho level significantly correlated with the 1,25-dihydroxyvitamin D level and inversely correlated with the parathyroid hormone level and the fractional excretion of phosphate. There were significant decreases in serum Klotho in patients with arterial stiffness defined as baPWV≥1400 cm/sec, atherosclerosis defined as maximum IMT≥1.1 mm and vascular calcification scores of ACI>0%. The serum Klotho level was a significant determinant of arterial stiffness, but not endothelial dysfunction, atherosclerosis or vascular calcification, in the multivariate analysis in either metabolic model, the CKD model or the CKD-MBD model. The adjusted odds ratio of serum Klotho for the baPWV was 0.60 (p = 0.0075).

Conclusions

Decreases in the serum soluble Klotho levels are independently associated with signs of vascular dysfunction such as arterial stiffness in patients with CKD. Further research exploring whether therapeutic approaches to maintain or elevate the Klotho level could improve arterial stiffness in CKD patients is warranted.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl₂. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.     

Chronic exposure to an extremely low‐frequency magnetic field induces depression‐like behavior and corticosterone secretion without enhancement of the hypothalamic–pituitary–adrenal axis in mice

An extremely low‐frequency magnetic field (ELF‐MF) is generated by power lines and household electrical devices. Many studies have suggested an association between chronic ELF‐MF exposure and anxiety and/or depression. The mechanism of these effects is assumed to be a stress response induced by ELF‐MF exposure. However, this mechanism remains controversial. In the present study, we investigated whether chronic ELF‐MF exposure (intensity, 3 mT; total exposure, 200 h) affected emotional behavior and corticosterone synthesis in mice. ELF‐MF‐treated mice showed a significant increase in total immobility time in a forced swim test and showed latency to enter the light box in a light–dark transition test, compared with sham‐treated (control) mice. Corticosterone secretion was significantly high in the ELF‐MF‐exposed mice; however, no changes were observed in the amount of the adrenocorticotropic hormone and the expression of genes related to stress response. Quantification of the mRNA levels of adrenal corticosteroid synthesis enzymes revealed a significant reduction in Cyp17a1 mRNA in the ELF‐MF‐exposed mice. Our findings suggest the possibility that high intensity and chronic exposure to ELF‐MF induces an increase in corticosterone secretion, along with depression‐ and/or anxiety‐like behavior, without enhancement of the hypothalamic–pituitary–adrenal axis. Bioelectromagnetics 34:43–51, 2013. © 2012 Wiley Periodicals, Inc.