A developmentally regulated hydroxyproline-rich glycoprotein in maize pericarp cell walls

We have studied the accumulation of peptidyl hydroxyproline in the pericarp of developing maize (Zea mays L., Golden cross Bantam sweet corn) kernels. Although this hydroxyproline accumulates throughout development, it is most soluble and its content per milligram dry weight greatest at midmaturation stages of development. Salt-soluble proteins containing this hydroxyproline from isolated cell walls of developing kernels were fractionated on a CsCl density gradient and on a Chromatofocusing column, resulting in the purification of an hydroxyproline-rich glycoprotein, PC-1. PC-1 is a basic protein of approximately 65 to 70 kilodaltons in molecular weight with an isoelectric point of at least 10.2 and a density of 1.38 to 1.39 in CsCl. Amino acid composition data indicate that it is rich in hydroxyproline, threonine, proline, lysine, and glycine. Its relation to dicot extensin is discussed.     

Cause for dark, chilling-induced inactivation of photosynthetic oxygen-evolving system in cucumber leaves

Effects on oxygen evolution of the storage of detached cucumber (Cucumis sativus) leaves at 0°C in the dark were investigated with thylakoids and oxygen-evolving photosystem II membranes isolated from stored leaves. The cold and dark treatment of leaves selectively inactivated electron transport on the oxidizing side of photosystem II. Photosystem II membranes isolated from treated leaves were largely depleted of two proteins of 20 and 14 kilodaltons, which correspond to the extrinsic 23- and 17- kilodalton proteins of spinach functioning in oxygen evolution. The manganese content of photosystem II membranes was also markedly reduced by the treatment. Thus, the inactivation of oxygen evolution induced by the dark, chilling treatment is ascribed to solubilization of the 20- and 14-kilodalton proteins and extraction of manganese.     

Kinetics of retroviral production from the amphotropic ΨCRIP murine producer cell line

Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/CRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/CRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.     

Short-term propamocarb exposure induces hepatic metabolism disorder associated with gut microbiota dysbiosis in adult male zebrafish

Propamocarb (PM) is a pesticide that is widely used to protect cucumbers and other plants from downy mildew.Recently,some studies indicated that PM exposure had potential toxic effects in animals.In this study,adult male zebrafish were exposed to 100 and 1000 μg/l PM for 7 days to assess its effects on metabolism and the gut microbiota.We observed a significant decrease in triglyceride (TG) in the livers of zebrafish that were exposed to 1000 μg/l PM for 7 days.At the same time,some genes related to glycolysis and lipid metabolism in the livers of zebrafish,including hexokinase-1 (HK1),pyruvate kinase (PK),acyi-CoA oxidase (Aco),peroxisome proliferator activated receptor alpha (Ppar-α),apolipoprotein A-Ⅳ-like (Apo),Acetyl CoA carboxylase-1 (Acc1),diacylglycerol acyltransferase (Dgat),and fatty acid synthase (Fas),were also decreased significantly after PM exposure.Based on GC-MS metabolomics analysis,a total of 48 metabolites changed significantly in the 1000 μg/l PM treatment group in comparison with the control group.These altered metabolites were mainly associated with the glycolysis,amino acid metabolism,and lipid metabolism pathways.Interestingly,we further found that the 1000 μg/l PM treatment group also showed significant elevations in Proteobacteria,Bacteroidetes,and Firmicutes at the phylum level.Sequencing of the 16S rRNA gene in the V3-V4 region also showed a significant change in the abundance and diversity of the gut microbiota in the 1000 μg/l PM treatment group.Our results indicated that exposure to PM for a short time could induce hepatic metabolic disorders and gut microbiota dysbiosis in adult male zebrafish.     

Induction and preliminary characterization of a novel halophage SNJ1 from lysogenic Natrinema sp. F5

Halophage SNJ1 was induced with mitomycin C from Natrinema sp. strain F5. The phage produces plaques on Natrinema sp. strain J7 only. The phage has a head of about 67 nm in diameter and a tail of 570 nm in length and belongs morphologically to the family Siphoviridae. The phage is strongly salt dependent; NaCl concentration affects the integrity of SNJ1, phage adsorption, and plaque formation. The optimal NaCl concentration for phage adsorption and plaque formation is 30% and 25%, respectively.     

1,25-dihydroxyvitamin D3 induces biphasic NF-kappaB responses during HL-60 leukemia cells differentiation through protein induction and PI3K/Akt-dependent phosphorylation/degradation of IkappaB

1,25-dihydroxyvitamin D(3) (VD(3)) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-kappaB) activity. Here we report a time-dependent biphasic regulation of NF-kappaB in VD(3)-treated HL-60 leukemia cells. After VD(3) treatment there was an early approximately 4 h suppression and a late 8-72 h prolonged reactivation of NF-kappaB. The reactivation of NF-kappaB was concomitant with increased IKK activities, IKK-mediated IkappaBalpha phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-kappaB/vitamin D responsive element promoters. In parallel with NF-kappaB stimulation, there was an up-regulation of NF-kappaB controlled inflammatory and anti-apoptotic genes such as TNFalpha, IL-1beta and Bcl-xL. VD(3)-triggered reactivation of NF-kappaB was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD(3)-stimulated IkappaBalpha phosphorylation as well as NF-kappaB-controlled gene expression. The early approximately 4 h VD(3)-mediated NF-kappaB suppression coincided with a prolonged increase of IkappaBalpha protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-kappaB in VD(3)-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD(3)-mediated immune-regulation.