NCF1/2/4 Are Prognostic Biomarkers Related to the Immune Infiltration of Kidney Renal Clear Cell Carcinoma

Neutrophil cytoplasmic factor 1/2/4 (NCF1/2/4) belongs to the NADPH oxidase complex, which is a cytoplasmic component, and its polymorphism is the main factor related to autoimmune diseases, which is probably caused by the regulation of peroxide. They also play a role in tumor growth and metastasis. This research is aimed at evaluating the biological function and prognostic role of NCF1, NCF2, and NCF4 genes in kidney renal clear cell carcinoma (KIRC) by using multiple online bioinformatics website, including Oncomine, GEPIA, UALCAN, Kaplan–Meier Plotter, TIMER, TISIDB, cBioPortal, LinkedOmics, GeneMANIA, and DAVID databases. The mRNA levels of NCFs were higher in KIRC tissues than in normal tissues. The overexpression of NCFs was significantly correlated with advanced pathological grades and individual cancer stages in KIRC. Meanwhile, the expressions of NCFs played an important role in the tumorigenesis and progression of KIRC. Prognostic value analysis suggested that high transcription levels of NCF1/4 were associated with poor overall survival in KIRC patients. In addition, results from the LinkedOmics database showed that the KEGG pathway related to NCFs mainly focused on immune activation and immune regulation function. NCF genetic alterations, including copy number amplification, missense mutation, and deep deletion, could be found through the cBioPortal database. Further, NCF expression was significantly correlated with infiltration levels of various immune cells as well as immune signatures. Protein-protein interaction network and enrichment analysis of NCF1/2/4 in KIRC showed that NCF coexpressed genes mainly associated with diverse immune marker sets showed significance. Overall, these results indicated that NCFs could be prognostic biomarkers as well as effective targets for diagnosis in KIRC.

Germ plasm-related structures in marine medaka gametogenesis; novel sites of Vasa localization and the unique mechanism of germ plasm granule arising

SummaryGerm plasm, a cytoplasmic factor of germline cell differentiation, is suggested to be a perspective tool for in vitro meiotic differentiation. To discriminate between the: (1) germ plasm-related structures (GPRS) involved in meiosis triggering; and (2) GPRS involved in the germ plasm storage phase, we investigated gametogenesis in the marine medaka Oryzias melastigma. The GPRS of the mitosis-to-meiosis period are similar in males and females. In both sexes, five events typically occur: (1) turning of the primary Vasa-positive germ plasm granules into the Vasa-positive intermitochondrial cement (IMC); (2) aggregation of some mitochondria by IMC followed by arising of mitochondrial clusters; (3) intramitochondrial localization of IMC-originated Vasa; followed by (4) mitochondrial cluster degradation; and (5) intranuclear localization of Vasa followed by this protein entering the nuclei (gonial cells) and synaptonemal complexes (zygotene–pachytene meiotic cells). In post-zygotene/pachytene gametogenesis, the GPRS are sex specific; the Vasa-positive chromatoid bodies are found during spermatogenesis, but oogenesis is characterized by secondary arising of Vasa-positive germ plasm granules followed by secondary formation and degradation of mitochondrial clusters. A complex type of germ plasm generation, ‘the follicle cell assigned germ plasm formation’, was found in late oogenesis. The mechanisms discovered are recommended to be taken into account for possible reconstruction of those under in vitro conditions.

Effects of the microtubule nucleator Mto1 on chromosomal movement, DNA repair, and sister chromatid cohesion in fission yeast

Although the function of microtubules (MTs) in chromosomal segregation during mitosis is well characterized, much less is known about the role of MTs in chromosomal functions during interphase. In the fission yeast Schizosaccharomyces pombe, dynamic cytoplasmic MT bundles move chromosomes in an oscillatory manner during interphase via linkages through the nuclear envelope (NE) at the spindle pole body (SPB) and other sites. Mto1 is a cytoplasmic factor that mediates the nucleation and attachment of cytoplasmic MTs to the nucleus. Here, we test the function of these cytoplasmic MTs and Mto1 on DNA repair and recombination during interphase. We find that mto1Δ cells exhibit defects in DNA repair and homologous recombination (HR) and abnormal DNA repair factory dynamics. In these cells, sister chromatids are not properly paired, and binding of Rad21 cohesin subunit along chromosomal arms is reduced. Our findings suggest a model in which cytoplasmic MTs and Mto1 facilitate efficient DNA repair and HR by promoting dynamic chromosomal organization and cohesion in the nucleus.

Cytoplasmic-nuclear incompatibility between wild-isolates of Caenorhabditis nouraguensis

ABSTRACTHow species arise is a fundamental question in biology. Species can be defined as populations of interbreeding individuals that are reproductively isolated from other such populations. Therefore, understanding how reproductive barriers evolve between populations is essential for understanding the process of speciation. Hybrid incompatibility (e.g. hybrid sterility and lethality) is a common and strong reproductive barrier in nature, but few studies have molecularly identified its genetic basis. Here we report a lethal incompatibility between two wild-isolates of the nematode Caenorhabditis nouraguensis. Hybrid inviability results from the incompatibility between a maternally inherited cytoplasmic factor from each strain and a recessive nuclear locus from the other. We have excluded the possibility that maternally inherited endosymbiotic bacteria cause the incompatibility by treating both strains with tetracycline and show that hybrid death is unaffected. Furthermore, cytoplasmic-nuclear incompatibility commonly occurs between other wild-isolates, indicating that this is a significant reproductive barrier within C. nouraguensis. We hypothesize that the maternally inherited cytoplasmic factor is the mitochondrial genome and that mitochondrial dysfunction underlies hybrid death. This system has the potential to shed light on the dynamics of divergent mitochondrial-nuclear coevolution and its role in promoting speciation.

Morphometrical analysis of bone marrow metamyelocyte in pernicious anemia

Introduction In pernicious anemia besides the presence of megaloblasts in the bone marrow, changes in myeloid series were seen; being the most evident among the metamyelocyte. The aim of this study was to perform the quantification of metamyelocyte of the bone marrow in pernicious anemia. Material and methods Between 2000-2006 in the Clinic of Hematology-Nis, 68 patients with pernicious anemia were examined and 30 with dyspeptic syndrome (control group). The group of patients with pernicious anemia in relation to pathohistologic changes of gastric mucosa was divided into three sub-groups. Morphometrical analysis of metamyelocyte of the bone marrow was carried out by the application of the double netlike system (B100). The following parameters were used: relative surface, contour length, absolute surface of nucleus and cytoplasm, absolute contour nucleus and cytoplasm density, shaped nucleus and cytoplasmic factor and nuclear-cytoplasmatic ratio of meta- myelocytes. Results Relative surface, contour length, absolute surface and contour density of nucleus and cytoplasm of metamyelocytes increased simultaneously with the degree of atrophic gastritis. Shaped nucleus and cytoplasmic factor and nuclear-cytoplasmatic ratio of metamyelocytes decreased in all examined groups in relation to the control group. Conclusion Not only are bone marrow erythroid elements scoped with megaloblastic changes but the changes on the level of leukocyte cells as well. The result of this is the phenomena of giant metamyelocytes.

308MITOCHONDRIA RELOCATION, MICROTUBULE ASSEMBLING AND PARTHENOGENETIC DEVELOPMENTAL COMPETENCE OF PIG OOCYTES

Developmental competence of in vitro-produced porcine embryos appears to be limited by specific maternally inherited cytoplasmic factors. We previously reported a relationship between mitochondria distribution during IVM, energy status, and oocyte developmental ability after parthenogenetic activation. The aim of the present study was to investigate the timing of mitochondria relocation during meiosis and the possible relationship with cytoskeleton organization in high and low competence oocytes. To this purpose, homogeneous groups of oocytes were matured in vitro (IVM) with 25% or 0% porcine follicular fluid (pff) to obtain different cytoplasmic competence (high and low, respectively) but similar nuclear maturation (Brevini et al., 2003, Theriogenology, 59, 440). After maturation, oocytes were parthenogenetically activated and cultured as previously described by Grupen et al., 2002 (Mol. Reprod. Dev., 62, 387–396). Active mitochondria were stained with MitoTracker® Orange CMTM Ros (Molecular Probes, Leiden, The Netherlands) at GV, MI and MII meiotic stages. At the same time microtubule organization was determined by immuno-cytochemistry using an antibody raised against α-tubulin (Sigma, St. Louis, MO, USA). Meiotic stages were assessed with DAPI. Specimens were examined with a Leica TCS-NT confocal microscope through an equatorial optical section. Nuclear maturation rate was comparable in the two groups at the end of IVM (46h). Mitochondria relocation from the periphery to the center of the oocyte was evident as early as 20h IVM in the 25% pff group (high competence), while 0% pff oocytes (low competence) did not show any mitochondria relocation at this time point. In agreement with the literature, α-tubulin was not detectable in GV oocytes, while at the end of IVM, α-tubulin was associated with the DNA, forming the meiotic spindle both in high and low competence oocytes. However, oocytes in the 25% pff group displayed a cytoplasmic microtubular organization that co-localized with mitochondria at 20 to 28h IVM. Conversely, α-tubulin was not detected in the cytoplasm of 0% pff oocytes at the same time points and 71% of these oocytes did not undergo any mitochondria relocation at all by the end of IVM. Altogether the present results show that mitochondria relocation takes place at a well-defined time during IVM and is temporally associated with the formation of the microtubule mesh in the oocyte cytoplasm. Low-developmental-competence oocytes display an altered mitochondria distribution and microtubule arrangement or seem to lack the temporal coupling of the two phenomena. We speculate that a tightly linked timing of mitochondria relocation and cytoskeleton microtubule formation in the cytoplasm of the oocyte during IVM may represent a key cytoplasmic factor regulating pig embryo parthenogenetic development.

Heat Shock Protein 90-Independent Activation of Truncated Hepadnavirus Reverse Transcriptase

ABSTRACT The reverse transcriptase (RT) encoded by hepadnaviruses (hepatitis B viruses) is a multifunctional protein critical for several aspects of viral assembly and replication. Reverse transcription is triggered by the specific interaction between the RT and an RNA signal located on the viral pregenomic RNA, termed ε, and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and ε serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-ε interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its cochaperones, which associates with the RT and facilitates the folding of the RT into an active conformation. We now report that extensive truncation removing the entire C-terminal RNase H domain and part of the central RT domain could relieve this dependence on Hsp90 for RT folding such that the truncated RT variants could function in ε interaction and protein priming independently of Hsp90. The presence of certain nonionic or zwitterionic detergent was sufficient to establish and maintain the truncated RT proteins in an active, albeit labile, state. Furthermore, we were able to refold an RT truncation variant de novo after complete denaturation. In contrast, the full-length RT and also RT variants with less-extensive C-terminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-to-be-identified cytoplasmic factor(s) led to a dramatic suppression of the RT activities. These results have important implications for RT folding and conformational maturation, Hsp90 chaperone function, and potential inhibition of RT functions by host cell factors.

Modulation of the Nuclear Factor κb Pathway by Shp-2 Tyrosine Phosphatase in Mediating the Induction of Interleukin (Il)-6 by IL-1 or Tumor Necrosis Factor

Shp-2, a src homology (SH)2-containing phosphotyrosine phosphatase, appears to be involved in cytoplasmic signaling downstream of a variety of cell surface receptors, although the mechanism is unclear. Here, we have determined a role of Shp-2 in the cytokine circuit for inflammatory and immune responses. Production of interleukin (IL)-6 in response to IL-1α or tumor necrosis factor (TNF)-α was nearly abolished in homozygous mutant (Shp-2−/−) fibroblast cells. The targeted Shp-2 mutation has no significant effect on the activation of the three types of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (Erk), c-Jun NH2-terminal kinase (Jnk), and p38, by IL-1/TNF, indicating that Shp-2 does not work through MAP kinase pathways in mediating IL-1/TNF-induced IL-6 synthesis. In contrast, IL-1/TNF-stimulated nuclear factor (NF)-κB DNA binding activity and inhibitor of κB (IκB) phosphorylation was dramatically decreased in Shp-2−/− cells, while the expression and activity of NF-κB–inducing kinase (NIK), Akt, and IκB kinase (IKK) were not changed. Reintroduction of a wild-type Shp-2 protein into Shp-2−/− cells rescued NF-κB activation and IL-6 production in response to IL-1/TNF stimulation. Furthermore, Shp-2 tyrosine phosphatase was detected in complexes with IKK as well as with IL-1 receptor. Thus, this SH2-containing enzyme is an important cytoplasmic factor required for efficient NF-κB activation. These results elucidate a novel mechanism of Shp-2 in cytokine signaling by specifically modulating the NF-κB pathway in a MAP kinase–independent fashion.