Involvement of Aberrant Glycosylation in Thyroid Cancer

Glycosylation is one of the most common posttranslational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharides structures are associated with many physiological and pathological events, including cell growth, migration and differentiation, and tumor invasion. Therefore, functional glycomics, which is a comprehensive study of the structures and functions of glycans, is attracting the increasing attention of scientists in various fields of life science. In cases of thyroid cancer, the biological characters and prognosis are completely different in each type of histopathology, and their oligosaccharide structures as well as the expression of glycosyltransferases are also different. In this review, we summarized our previous papers on oligosaccharides and thyroid cancers and discussed a possible function of oligosaccharides in the carcinogenesis in thyroid cancer

Horner's Rule-Based Multiplication over Fp and Fp^n: A Survey

International audienceThis paper aims at surveying multipliers based on Horner's rule for finite field arithmetic. We present a generic architecture based on five processing elements and introduce a classification of several algorithms based on our model. We provide the readers with a detailed description of each scheme which should allow them to write a VHDL description or a VHDL code generator

Site-specific and linkage analyses of fucosylated N-glycans on haptoglobin in sera of patients with various types of cancer: possible implication for the differential diagnosis of cancer: possible implication for the differential diagnosis of cancer

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported observed in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 in only cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers

3-Ketosteroid-Δ1 -dehydrogenase of Rhodococcus rhodochrous: Sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系The gene encoding 3-ketosteroid-Δ1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Δ1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Δ1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme

Establishment of an antibody specific for cancer-associated haptoglobin: a possible implication of clinical investigation

We previously found that the serum level of fucosylated haptoglobin (Fuc-Hpt) was significantly increased in pancreatic cancer patients. To delineate the mechanism underlying this increase and develop a simple detection method, we set out to generate a monoclonal antibody (mAb) specific for Fuc-Hpt. After multiple screenings by enzyme-linked immunosorbent assay (ELISA), a 10-7G mAb was identified as being highly specific for Fuc-Hpt generated in a cell line as well as for Hpt derived from a pancreatic cancer patient. As a result from affinity chromatography with 10-7G mAb, followed by lectin blot and mass spectrometry analyses, it was found that 10-7G mAb predominantly recognized both Fuc-Hpt and prohaptoglobin (proHpt), which was also fucosylated. In immunohistochemical analyses, hepatocytes surrounding metastasized cancer cells were stained by the 10-7G mAb, but neither the original cancer cells themselves nor normal hepatocytes exhibited positive staining, suggesting that metastasized cancer cells promote Fuc-Hpt production in adjacent hepatocytes. Serum level of Fuc-Hpt determined with newly developed ELISA system using the 10-7G mAb, was increased in patients of pancreatic and colorectal cancer. Interestingly, dramatic increases in Fuc-Hpt levels were observed at the stage IV of colorectal cancer. These results indicate that the 10-7G mAb developed is a promising antibody which recognizes Fuc-Hpt and could be a useful diagnostic tool for detecting liver metastasis of cancer.This study was performed as a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan and was supported by JSPS KAKENHI Grant Number JP16H05226

Terminal fucosylation of haptoglobin in cancer-derived exosomes during cholangiocarcinoma progression

BackgroundCholangiocarcinoma (CCA) is a silent tumor with a high mortality rate due to the difficulty of early diagnosis and prediction of recurrence even after timely surgery. Serologic cancer biomarkers have been used in clinical practice, but their low specificity and sensitivity have been problematic. In this study, we aimed to identify CCA-specific glycan epitopes that can be used for diagnosis and to elucidate the mechanisms by which glycosylation is altered with tumor progression.MethodsThe serum of patients with various cancers was fractioned into membrane-bound and soluble components using serial ultracentrifugation. Lectin blotting was conducted to evaluate glycosylation. Proteins having altered glycosylation were identified using proteomic analysis and further confirmed using immunoblotting analysis. We performed HPLC, gene analysis, real-time cargo tracking, and immunohistochemistry to determine the origin of CCA glycosylation and its underlying mechanisms. Extracellular vesicles (EV) were isolated from the sera of 62 patients with CCA at different clinical stages and inflammatory conditions and used for glycan analysis to assess their clinical significance.ResultsThe results reveal that glycosylation patterns between soluble and membrane-bound fractions differ significantly even when obtained from the same donor. Notably, glycans with α1-3/4 fucose and β1-6GlcNAc branched structures increase specifically in membrane-bound fractions of CCA. Mechanically, it is primarily due to β-haptoglobin (β-Hp) originating from CCA expressing fucosyltransferase-3/4 (FUT 3/4) and N-acetylglucosaminyltransferase-V (MGAT5). Newly synthesized β-Hp is loaded into EVs in early endosomes via a KFERQ-like motif and then secreted from CCA cells to induce tumor progression. In contrast, β-Hp expressed by hepatocytes is secreted in a soluble form that does not affect CCA progression. Moreover, evaluation of EV glycosylation in CCA patients shows that fucosylation level of EV-Hp gradually increases with tumor progression and decreases markedly when the tumors are eliminated by surgery.ConclusionThis study suggests that terminal fucosylation of Hp in cancer-derived exosomes can be a novel glycan marker for diagnosis and prognosis of CCA. These findings highlight the potential of glycan analysis in different fractions of serum for biomarker discover for other diseases. Further research is needed to understand the role of fucosylated EVs on CCA progression

エストロゲン受容体陽性乳がん細胞において、BIG3は複数のKaryopherin-αを介したPHB2のエストロゲン依存性核移行を阻害する

We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells

Palmitoylated ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi. In this study, we show that active palmitoylated Ras proteins mainly localize intracellularly at REs and that REs act as a way station along the post-Golgi exocytic pathway to the PM. H-Ras requires two palmitoyl groups for RE targeting. The lack of either or both palmitoyl groups leads to the mislocalization of the mutant proteins to the endoplasmic reticulum, Golgi apparatus, or the PM. Therefore, we demonstrate that palmitoylation directs Ras proteins to the correct intracellular organelles for trafficking and activity. © 2010 Misaki et al

Vibrio fluvialis in Patients with Diarrhea, Kolkata, India

We identified 131 strains of Vibrio fluvialis among 400 nonagglutinating Vibrio spp. isolated from patients with diarrhea in Kolkata, India. For 43 patients, V. fluvialis was the sole pathogen identified. Most strains harbored genes encoding hemolysin and metalloprotease; this finding may contribute to understanding of the pathogenicity of V. fluvialis