Diversity of Ca2+-induced morphology revealed by morphological phenotyping of Ca2+-sensitive mutants of Saccharomyces cerevisiae

Yeast cell morphology can be treated as a quantitative trait using the image processing software CalMorph. In the present study, we investigated Ca(2+)-induced morphological changes in Ca(2+)-sensitive (cls) mutants of Saccharomyces cerevisiae, based on the discovery that the characteristic Ca(2+)-induced morphological changes in the Ca(2+)-sensitive mutant zds1 reflect changes in the Ca(2+) signaling-mediated cell cycle control pathway. By applying hierarchical cluster analysis to the quantitative morphological data of 58 cls mutants, 31 of these mutants were classified into seven classes based on morphological similarities. The patterns of morphological change induced by Ca(2+) in one class differed from those of another class. Based on the results obtained using versatile methods for phenotypic analysis, we conclude that a high concentration of Ca(2+) exerts a wide variety of effects on yeast and that there are multiple Ca(2+)-regulatory pathways that are distinct from the Zds1p-related pathway.     

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H₂O₂ exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H₂O₂ exposure. The present work shows that this tick cell line could tolerate high H₂O₂ concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

A factor of inducing IgE from a filarial parasite is an agonist of human CD40

Immune responses to parasitic helminth are usually characterized by quite mysterious phenomena: dominance of Th2-like immunity and antigen-nonspecific IgE secretion. We previously purified a factor from Dirofilaria immitis that induces antigen-nonspecific IgE in rats and named it DiAg. In the presence of IL-4, DiAg induces mouse B cells to secrete IgE, which is antigen-nonspecific polyclonal antibody. We investigated the biochemical characteristics of DiAg as a factor of inducing IgE in this study. Recombinant DiAg (rDiAg) with interleukin (IL)-4 induced IgE synthesis in highly purified human normal B cells in vitro cell culture systems. The addition of recombinant human soluble CD40 IgG fusion protein (rsCD40-Ig) inhibited induction of IgE synthesis by rDiAg with IL-4. Monocyte cells were stimulated with rDiAg and recombinant human soluble CD40L (rsCD40L); IL-12 and TNF-alpha were induced. The addition of rsCD40-Ig to THP-1 cells activated with rDiAg and rsCD40L inhibited the production of IL-12. rDiAg bound to the monocyte cell membrane fraction and recombinant human soluble CD40; this binding of rDiAg was competitively inhibited by addition of rsCD40L. Moreover, in CD40-deficient mice, IgE production and MLN-B cell proliferation by rDiAg were completely absent. Based on these results, we concluded that DiAg is an agonist of CD40.     

Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci

We determined the expression and subcellular localization of nuclear protein NP95 during the cell cycle in mouse 3T3 cells. The levels of NP95 mRNA and protein were extremely low in quiescent cells; however, stimulation with 10% serum increased their expressions in a time course similar to that of the late growth-regulated gene proliferating cell nuclear antigen (PCNA). Subnuclear location of NP95 dynamically changed during the cell cycle. Double immunostaining for NP95 and chromatin-bound PCNA, a marker of DNA replication sites, revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. Distinct localization of the two proteins, however, became evident in mid-S phase, and thereafter, many chromatin-bound PCNA foci not carrying NP95 foci could be detected. In G2 phase, nodular NP95 foci were still identified without any chromatin-bound PCNA foci. Chromatin-bound PCNA was observed as a pre-DNA replication complex at the G1/S boundary synchronized by hydroxyurea treatment, while NP95 was detected in nucleolar regions as unique large foci. There was no significant redistribution of NP95 foci shortly after DNA damage by gamma-irradiation. Nodular NP95 foci characteristically seen in G2 phase were also detected in G2-arrested cells following gamma-irradiation. Taken together, our results indicate that NP95 is assigned to a late growth-regulated gene and suggest that NP95 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events.     

Organ-specific pancreatic tumor growth properties and tumor immunity

We established a model of orthotopic injection of a syngeneic pancreatic tumor cell line in C57BL/6 mice and evaluated the effects of organ site on induction of immunity to a tumor-specific antigen, MUC1. Mice were challenged with a syngeneic pancreatic adenocarcinoma cell line that expressed MUC1 (Panc02-MUC1) by orthotopic injection into the pancreas, or by subcutaneous injection. Tumor cells injected into the pancreas grew much faster than those injected subcutaneously. Mice challenged subcutaneously with Panc02-MUC1 rejected tumors or developed slowly growing tumors that were negative for MUC1 expression. In contrast, mice challenged orthotopically into the pancreas developed progressive tumors that were positive for MUC1 expression. Sera from mice that rejected Panc02-MUC1 (tumor-immune mice) showed no detectable IgG1 and IgM titers against the MUC1 tandem-repeat peptide, whereas mice with progressive tumor growth had significant titers of IgG1 and IgM specific for MUC1. This suggests that the humoral immune response was ineffective in mediating tumor rejection. The results show that the growth properties and immunological rejection of pancreatic tumors is affected by the organ site at which the tumor grows. Received: 25 April 1998 / Accepted: 7 October 1998     

Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT)

Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1alpha (HIF-1alpha) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between -165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.