Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.     

Designed and real working situations in machine systems operation.

New work situations designed at the stage when new machine systems are introduced are realized on the assumption that the new systems can maintain their designed functions consistently, generally eliminating previous work habits and without sufficient knowledge about real working processes and skills. This may produce differences between designed and real working situations. Some examples are presented from observations on influence of modern design of cargo ships on their crews. It was difficult for crews to maintain stable working conditions, especially when machine systems deviated from their designed functions. Often the crew had to work in off-duty hours giving up private freetime activities. Among various factors contributing to the discrepancies between designed and real work, lack of availability of the new systems is the most important factor. Also important is lack of back-up systems which would function either when the machine systems are out of order or when previous working skills and habits must be applied. A need for developing methods of evaluation of these two factors from ergonomic points of view is pointed out.     

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants’ genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites’ drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.     

Epstein-Barr virus nuclear antigen 3C (EBNA3C) interacts with the metabolism sensing C-terminal binding protein (CtBP) repressor to upregulate host genes

Epstein-Barr virus (EBV) infection is associated with the development of specific types of lymphoma and some epithelial cancers. EBV infection of resting B-lymphocytes in vitro drives them to proliferate as lymphoblastoid cell lines (LCLs) and serves as a model for studying EBV lymphomagenesis. EBV nuclear antigen 3C (EBNA3C) is one of the genes required for LCL growth and previous work has suggested that suppression of the CDKN2A encoded tumor suppressor p16^INK4A and possibly p14^ARF is central to EBNA3C’s role in this growth transformation. To directly assess whether loss of p16 and/or p14 was sufficient to explain EBNA3C growth effects, we used CRISPR/Cas9 to disrupt specific CDKN2A exons in EBV transformed LCLs. Disruption of p16 specific exon 1α and the p16/p14 shared exon 2 were each sufficient to restore growth in the absence of EBNA3C. Using EBNA3C conditional LCLs knocked out for either exon 1α or 2, we identified EBNA3C induced and repressed genes. By trans-complementing with EBNA3C mutants, we determined specific genes that require EBNA3C interaction with RBPJ or CtBP for their regulation. Unexpectedly, interaction with the CtBP repressor was required not only for repression, but also for EBNA3C induction of many host genes. Contrary to previously proposed models, we found that EBNA3C does not recruit CtBP to the promoters of these genes. Instead, our results suggest that CtBP is bound to these promoters in the absence of EBNA3C and that EBNA3C interaction with CtBP interferes with the repressive function of CtBP, leading to EBNA3C mediated upregulation.     

MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.     

A Novel Cell-Sheet Technology That Achieves Durable Factor VIII Delivery in a Mouse Model of Hemophilia A

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.     

Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes

Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.