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Rabbit anti-asialo-GM1 (ASGM1) serum or polyclonal antibodies can eliminate mouse splenic natural killer (NK) cell activity in vitro and in vivo. We developed rabbit monoclonal antibodies (mAbs) against ASGM1 using a single-cell analysis and isolation system. Five mAbs (GA109, GA115, GA116, GA131, and GA134) that were reactive to ASGM1 were isolated from the spleen lymphocytes of rabbits immunized with ASGM1. Enzyme-linked immunosorbent assay and thin-layer chromatography immunostaining results showed that the mAbs strongly reacted with ASGM1. Two mAbs (GA116 and GA134) reacted exclusively with ASGM1, whereas three mAbs (GA109, GA115, and GA131) showed slight or considerable cross-reactivity with GM1. The administration of the mAbs (4–20 μg) to BALB/c mice completely abolished NK cell activity in vivo. The anti-ASGM1 rabbit mAbs obtained in this study may provide a useful and reproducible tool for various future studies, such as depleting NK cell activity to enhance xenograft engraftment in mouse models.

Rabbit anti-asialo-GM1 (ASGM1) serum or polyclonal antibodies can eliminate mouse splenic natural killer (NK) cell activity in vitro and in vivo. We developed rabbit monoclonal antibodies (mAbs) against ASGM1 using a single-cell analysis and isolation system. Five mAbs (GA109, GA115, GA116, GA131, and GA134) that were reactive to ASGM1 were isolated from the spleen lymphocytes of rabbits immunized with ASGM1. Enzyme-linked immunosorbent assay and thin-layer chromatography immunostaining results showed that the mAbs strongly reacted with ASGM1. Two mAbs (GA116 and GA134) reacted exclusively with ASGM1, whereas three mAbs (GA109, GA115, and GA131) showed slight or considerable cross-reactivity with GM1. The administration of the mAbs (4-20 μg) to BALB/c mice completely abolished NK cell activity in vivo. The anti-ASGM1 rabbit mAbs obtained in this study may provide a useful and reproducible tool for various future studies, such as depleting NK cell activity to enhance xenograft engraftment in mouse models.

Ras gene mutations are frequently observed in many types of cancers. However, there are currently no effective anticancer drugs against Ras-induced cancers. Therefore, identifying the downstream effectors of the Ras signaling pathway can facilitate the development of promising novel therapeutic approaches. We previously showed that oncogenic Ras induces the expression of the receptor tyrosine kinase c-Mer proto-oncogene tyrosine kinase (MerTK) in an interleukin-1 family member NF-HEV/IL-33-dependent manner and that IL-33 and MerTK contribute to oncogenic Ras-induced cell migration. In the present study, we purified the MerTK complex from NIH-3T3 cells transformed by the expression of oncogenic Ras, H-Ras (G12V). Mass spectrometric analysis identified STK38 (also known as NDR1) as a candidate binding partner for MerTK. STK38 is a serine/threonine protein kinase that plays diverse roles in normal and cancerous cells. In addition to MerTK knockdown, STK38 knockdown effectively attenuated the H-Ras (G12V)-induced migration of NIH-3T3 cells. STK38 kinase activity is required for oncogenic Ras-induced cell migration and MerTK tyrosine phosphorylation. Furthermore, MerTK or STK38 knockdown attenuated the activation of Rac1 and Cdc42. Taken together, these results revealed a novel role for STK38 in oncogenic Ras-induced enhanced cell migration, which may be useful for developing novel therapeutic strategies targeting Ras-mutated cells.

Chronic myeloid leukemia (CML) is induced by the expression of the fused tyrosine kinase BCR-ABL, which is caused by a chromosomal translocation. BCR-ABL inhibitors have been used to treat CML; however, the acquisition of resistance by CML cells during treatment is a serious issue. We herein demonstrated that BCR-ABL induced the expression of the RNA helicase DDX5 in K562 cells derived from CML patients in a manner that was dependent on its kinase activity, which resulted in cell proliferation and survival. The knockout of DDX5 decreased the expression of BIRC5 (survivin) and activated caspase 3, leading to apoptosis in K562 cells. Similar results were obtained in cells treated with FL118, an inhibitor of DDX5 and a derivative compound of camptothecin (CPT). Furthermore, FL118 potently induced apoptosis not only in Ba/F3 cells expressing BCR-ABL, but also in those expressing the BCR-ABL T315I mutant, which is resistant to BCR-ABL inhibitors. Collectively, these results revealed that DDX5 is a critical therapeutic target in CML and that FL118 is an effective candidate compound for the treatment of BCR-ABL inhibitor-resistant CML.

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

Ildr2 was initially identified as a genetic modifier of diabetes susceptibility in B6.DBA  Lep ob  congenic mice, and was associated with decreased β-cell replication rates, reduced β-cell mass, and persistent mild hypoinsulinemic hyperglycemia. However, the molecular mechanisms of how the ILDR2 protein is involved in these effects are largely unknown. We sought to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underpinning ILDR2 function in pancreatic β-cells. Using TAP tag technology, we purified proteins interacting with ILDR2 in the pancreatic β-cell line MIN6, and identified the endoplasmic reticulum resident chaperones, GRP78 and PDIA1, as novel proteins interacting with ILDR2. We demonstrated that GRP78 interacted with ILDR2 and was possibly involved in ILDR2 stabilization by inhibiting ubiquitin–proteasome degradation. Additionally, adenoviral ILDR2 knockdown led to reduced glucose-responsive insulin secretion in MIN6 β-cells, suggesting ILDR2 may be implicated in a new pathway in hypoinsulinemic hyperglycemia. These data provide evidence for a novel association between GRP78 and ILDR2, and suggest GPR78-ILDR2 may a novel target for diabetic therapeutic modulation in decreased insulin secretion.

Background Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1 , which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic ‘Hamlin’ sweet orange by antisense-overexpression. Results The candidate gene CitRKD1 , predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain-containing protein. CitRKD1 of satsuma mandarin comprised two alleles ( CitRKD1-mg1 and CitRKD1-mg2 ) at the polyembryonic locus controlling embryonic type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T 1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds. Conclusion CitRKD1 at the polyembryonic locus plays a principal role in regulating citrus somatic embryogenesis. CitRKD1 comprised multiple alleles that were divided into two types, polyembryonic alleles with a MITE insertion in the upstream region and monoembryonic alleles without it. CitRKD1 was transcribed in reproductive tissues of polyembryonic varieties with the polyembryonic allele. The MITE insertion in the upstream region of CitRKD1 might be involved in regulating the transcription of CitRKD1 .

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12- O -tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

In citrus breeding programs, male sterility is an important trait for developing seedless varieties. Sterility associated with the male sterile cytoplasm of Kishu mandarin (Kishu-cytoplasm) has been proposed to fit the cytoplasmic male sterility (CMS) model. However, it remains undetermined whether CMS in citrus is controlled by interactions between sterile cytoplasm and nuclear restorer-of-fertility ( Rf ) genes. Accordingly, mechanisms underlying the control of the wide phenotypic variation in pollen number for breeding germplasm should be elucidated. This study aimed to identify complete linkage DNA markers responsible for male sterility at the MS-P1 region based on fine mapping. Two P-class pentatricopeptide repeat (PPR) family genes were identified as candidates for Rf based on predicted mitochondrial localization and higher expression in a male fertile variety/selected strain than in a male sterile variety. Eleven haplotypes (HT1–HT11) at the MS-P1 region were defined based on genotyping of DNA markers. Association analysis of diplotypes at the MS-P1 region and the number of pollen grains per anther (NPG) in breeding germplasms harboring Kishu-cytoplasm revealed that the diplotypes in this region influenced NPG. Among these haplotypes, HT1 is a non-functional restorer-of-fertility ( rf ) haplotype; HT2, a less-functional Rf ; HT3–HT5 are semi-functional Rfs ; and HT6 and HT7 are functional Rfs . However, the rare haplotypes HT8–HT11 could not be characterized. Therefore, P-class PPR family genes in the MS-P1 region may constitute the nuclear Rf genes within the CMS model, and a combination of the seven haplotypes could contribute to phenotypic variation in the NPG of breeding germplasms. These findings reveal the genomic mechanisms of CMS in citrus and will contribute to seedless citrus breeding programs by selecting candidate seedless seedlings using the DNA markers at the MS-P1 region.

[This corrects the article DOI: 10.3389/fpls.2023.1163358.].