Clinic implication of MUC1 O-glycosylation and C1GALT1 in esophagus squamous cell carcinoma

Esophagus squamous cell carcinoma(ESCC) is one of the most aggressive malignant tumors in the world. Our previous data demonstrates that oncoprotein MUC1 is related with metastasis and poor outcome of ESCC. However, alteration of MUC1 in ESCC remains unclear. Using ONCOMINE and COSMIC databases, we analyzed MUC1 gene copy numbers and gene mutations and found that MUC1 had high expression level but few gene mutations in ESCC. Further study of ESCC samples indicated that MUC1 O-glycosylation levels were higher in tumor tissues than that in para-carcinoma tissues in 10 of 14 pairs of ESCC samples. Moreover, we verified a potential link between MUC1 O-glycosylation and C1 GALT1, which was further supported by IHC analysis on 38 ESCC and 19 para-carcinoma samples. More importantly, co-expression of MUC1 Oglycosylation and C1 GALT1 presented positive correlations with both lymph node metastasis and survival time of ESCC patients. Our work collectively indicates that C1 GALT1 is associated with O-glycosylated MUC1 in ESCC, not only suggesting a diagnostic significance of C1 GALT1 and MUC1 O-glycosylation in ESCC, but also opening novel insights into targeting C1 GALT1 and MUC1 O-glycosylation to suppress ESCC cells metastasis in patients.     

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible <Emphasis Type="Italic">N</Emphasis>- and <Emphasis Type="Italic">O</Emphasis>-linked glycosylation

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and β-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.     

Prognostic significance of mucin expression profiles in breast carcinoma with signet ring cells: a clinicopathological study

Background

Signet ring cells (SRCs) often accompany gastrointestinal carcinoma, referred to as SRC carcinoma; however, breast cancers containing SRCs have not been well characterized, leaving the prognostic significance of SRCs undetermined. We have described clinicopathological characteristics of patients with breast cancer containing SRCs in relation to the expression levels of MUC1, MUC2, MUC4, MUC5AC, and MUC6.

Methods

Twenty-two breast cancer cases with variable degrees of SRC population were retrospectively studied. Each case was categorized as high (>31 %) or low (<30 %) SRC tumor. The SRCs were morphologically classified into the intra-cytoplasmic lumen (ICL) type, or the non-ICL type. The expression levels of MUC1, MUC2, MUC4, MUC5AC and MUC6 were determined immunohistochemically. Depending on its subcellular localization, MUC1 was categorized as the luminal and cytoplasmic (LC) type, or the cytoplasmic with circumferential membranous accentuation (CM) type. These histological findings were compared with other clinicopathological parameters.

Results

The series consisted of invasive ductal carcinoma (n?=?9), invasive lobular carcinoma (n?=?9), and mucinous carcinoma (n?=?4) cases. The SRC population accounted for 8–81 % of the tumor cells. Eight cases had ICL type SRCs, and the remaining 14 had non-ICL type SRCs. Neither the high (n?=?12) and low (n?=?10) percentage of SRCs, nor the SRC types affected the clinicopathological parameters. In the low MUC1 group (n?=?11), larger tumors, higher nuclear grade, lymph node metastasis, and negativity for estrogen receptor was more frequently identified compared to the high MUC1 group (n?=?11; p?=?0.01, p?=?0.002, p?=?0.008, and p?=?0.02, respectively). The CM group (n?=?7) had more patients with large-sized tumors, lymph node metastasis, lymphovascular invasion, and higher Ki67 indices than the LC group (n?=?15; p?=?0.04, p?=?0.001, p?=?0.006, and p?=?0.03, respectively). The expression levels of MUC2, MUC4, MUC5AC, and MUC6 showed no clinicopathological significance. Two patients with low MUC1 expression and CM patterns had tumor recurrence, resulting in death, while all the other patients survived without recurrence.

Conclusion

Our results demonstrate that in breast cancers containing SRCs, low MUC1 expression and/or its CM subcellular localization patterns are associated with unfavorable clinicopathological factors. The utility of MUC1 expression as a prognostic marker remains to be verified in future studies.

     

Tandem duplications of receptor-like kinase genes reshaped the homologous chromosome 1 regions in <Emphasis Type="Italic">Oryza sativa</Emphasis> ssp. <Emphasis Type="Italic">indica</Emphasis> and <Emphasis Type="Italic">japonica</Emphasis> and their possible ancestral genomes

Plant receptor-like kinase (Rlk) genes form a large family, each encoding a protein with a signal motif, a single transmembrane region, and a cytoplasmic kinase domain. Various gene duplications have contributed to the establishment and expansion of the family. Here, we characterized the formation and evolution of the Rlk gene family in cultivated rice and their possible progenitors. Using wheat Rlk gene sequences, we identified orthologs from the genomes of domesticated rice subspecies Oryza sativa ssp. japonica and ssp. indica and their putative progenitors O. glaberrima and O. rufipogon. The four chromosome 1 orthologous regions ranged from 103 to 281 kb comprising 181 syntenic blocks with 75 to 100% sequence identity. These regions contained 11–19 Triticum aestivum kinases (Taks) and 10–15 Lr10 receptor-like kinases (Lrks) organized in clusters and 3–12 transposable elements (TEs). Dot plot analyses showed that the 4 regions had 21–37 conserved catalytic domains, mainly in protein kinases (PKs) and tyrosine kinases (TyrKs) in coupling state. Over 50% of the sequences of glaberrima/rufipogon and japonica/indica pairs were colinear, while japonica/indica displayed a marked sequence expansion with duplicated genes and TEs. A total of 2312 single nucleotide polymorphisms (SNPs) and insertion-deletions (INDELs) were identified between japonica and indica. Duplication of the Rlk genes in O. glaberrima and O. rufipogon occurred after the grass species radiation and before the divergence of O. rufipogon from O. glaberrima; the orthologous Rlk genes from O. japonica and O. indica duplicated after O. sativa separated from O. rufipogon; paralogs, obtained through extensive duplication, happened after the separation of rice from maize. Tandem duplication was the major factor contributing to the gene copy number variation and genome size expansion.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Association between the length of the <Emphasis Type="Italic">MUC8-</Emphasis>minisatellite 5 region and susceptibility to chronic obstructive pulmonary disease (COPD)

In asthma and chronic obstructive pulmonary disease (COPD), mucins display disease-related alterations caused by airway mucus obstruction. MUC5AC, MUC5B and MUC8 are known as the major secretory mucins in human airway epithelial cells. Analysis of mucin genes has identified the presence of several features with a variable number of tandem repeats (VNTR; minisatellites) in the central region of each mucin. In our previous study, six minisatellites in the region of the MUC8 gene were identified, and the MUC8-MS5 minisatellite showed the highest heterozygosity among them. In this study, we evaluated the relationship between MUC8-MS5 and susceptibility to asthma and COPD. A case-control study was performed with 229 controls, 123 COPD cases and 77 asthma cases. A significant association (OR 3.96) between short alleles (2/2 repeats) and the occurrence of COPD was observed [95% confidence interval (CI) 1.32–11.88; p?=?0.008]. Hence, the increased frequency of 2/2 homo-short alleles were also found in asthma cases (3.11; CI 0.88–11.05; p?=?0.066), though this association was not statistically significant. These results revealed a genetic association between MUC8 and COPD, and that the specific short minisatellite alleles (2/2) of MUC8-MS5 may be a risk factor for COPD.     

In planta characterization of a tau class glutathione S-transferase gene from <Emphasis Type="Italic">Juglans regia</Emphasis> (<Emphasis Type="Italic">JrGSTTau1</Emphasis>) involved in chilling tolerance

Key message

JrGSTTau1 is an important candidate gene for plant chilling tolerance regulation.

Abstract

A tau subfamily glutathione S-transferase (GST) gene from Juglans regia (JrGSTTau1, GeneBank No.: KT351091) was cloned and functionally characterized. JrGSTTau1 was induced by 16, 12, 10, 8, and 6 °C stresses. The transiently transformed J. regia showed much greater GST, glutathione peroxidase (GPX), superoxide dismutase (SOD), and peroxidase (POD) activities and lower H₂O₂, malondialdehyde (MDA), reactive oxygen species (ROS), and electrolyte leakage (EL) rate than prokII (empty vector control) and RNAi::JrGSTTau1 under cold stress, indicating that JrGSTTau1 may be involved in chilling tolerance. To further confirm the role of JrGSTTau1, JrGSTTau1 was heterologously expressed in tobacco, transgenic Line5, Line9, and Line12 were chosen for analysis. The germinations of WT, Line5, Line9, and Line12 were similar, but the fresh weight, primary root length, and total chlorophyll content (tcc) of the transgenic lines were significantly higher than those of WT under cold stress. When cultivated in soil, the GST and SOD activities of transgenic tobacco were significantly higher than those of WT; however, the MDA and H₂O₂ contents of WT were on average 1.47- and 1.96-fold higher than those of Line5, Line9, and Line12 under 16 °C. The DAB, Evans blue, and PI staining further confirmed these results. Furthermore, the abundances of NtGST, MnSOD, NtMAPK9, and CDPK15 were elevated in 35S::JrGSTTau1 tobacco compared with WT. These results suggested that JrGSTTau1 improves the plant chilling tolerance involved in protecting enzymes, ROS scavenging, and stress-related genes, indicating that JrGSTTau1 is a candidate gene for the potential application in molecular breeding to enhance plant abiotic stress tolerance.

     

Mutant <Emphasis Type="Italic">TP53</Emphasis> G245C and R273H promote cellular malignancy in esophageal squamous cell carcinoma

Background

TP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102–292) and mainly reside in six “hotspot” residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53?/?.

Results

MTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.

Conclusions

These results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

     

Nucleotide Diversity of the Coding and Promoter Regions of <Emphasis Type="Italic">DREB1D</Emphasis>, a Candidate Gene for Drought Tolerance in <Emphasis Type="Italic">Coffea</Emphasis> Species

Climate change is posing a major challenge to coffee production worldwide leading to a need for the development of coffee cultivars with increased drought tolerance. In several plant species, the use of DREB genes in crop improvement has achieved promising results to desiccation tolerance engineering. Recent studies reported CcDREB1D specific patterns of expression in Coffea canephora and functional evidence of this gene involvement in drought stress responses. However, knowledge on natural diversity of this gene is largely unknown. In this context, this study aimed at evaluating the sequence variability of the DREB1D gene in several Coffea genotypes. Nucleotide variation in promoters and coding regions of this gene were evaluated in a population consisting of 38 genotypes of C. canephora, C. arabica and C. eugenioides, most of them characterized by different phenotypes (tolerance vs. susceptibility) in relation to drought. The genetic diversity of the loci revealed different haplotypes for the promoter and coding regions. In particular, our findings suggest association between drought tolerance and the genetic variations on DREB1D promoter regions, but not with those from its corresponding coding regions. Gene expression studies revealed up-regulated expression of DREB1D gene upon drought mainly in leaves of drought-tolerant clones of C. canephora, and in response to drought, high, and low temperatures in leaves of C. arabica, suggesting a key role of this gene in coffee responses to abiotic stress.     

Polymorphism in promoter regions of matrix metalloproteinases (<Emphasis Type="Italic">MMP1</Emphasis>, <Emphasis Type="Italic">MMP9</Emphasis>, and <Emphasis Type="Italic">MMP12</Emphasis>) in chronic obstructive pulmonary disease patients

Our studies have shown that the genotype and allele frequencies of polymorphisms G(?1607)GG of MMP1 gene, C(?1562)T of MMP9 gene, and A(?82)G of MMP12 gene do not significantly differ in the samples of chronic obstructive pulmonary disease (COPD) patients (N = 318) and healthy controls (N = 319) dwelling in Bashkortostan Republic. However, association of (?1562)T allele of the MMP9 gene with the severity of COPD disease progression has been revealed. In COPD patients at stage 4 of the disease, the frequency of allele T was significantly higher that in patients with the stages 2 and 3 (15.89% versus 8.38%; χ² = 7.804; d.f. = 1; P = 0.005; OR = 2.06 95% CI 1.22–3.49). The distribution of the genotype frequencies of C(?1562)T polymorphism of MMP9 gene significantly differed between the patients with various COPD severity (χ² = 9.849; d.f. = 2; P = 0.007). The individuals with rare genotype TT were revealed only among patients with severe COPD form (3.97% versus 0%; χ² = 4.78; P = 0.029; P _cor = 0.058). Analysis of this polymorphism in patients with early COPD onset (younger than 55 years old) has shown a significant increase in the allele T frequency in the group of patients with severe COPD (stage 4 according to GOLD) compared to the patients of the same age but with less severe COPD progression (χ² = 5.26; d.f. = 1; P = 0.022). As the major clinical characteristics of stage 4 COPD is the development of pulmonary emphysema as well as bronchial walls deformation, we suggest that the increased expression of MMP9 gene caused by genetic polymorphism in the gene promoter is important in the early development of serious complications of the disease.     

Selection of reference genes for quantitative real-time PCR in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Requirement of the isocitrate lyase gene <Emphasis Type="Italic">ICL1</Emphasis> for <Emphasis Type="Italic">VPS41</Emphasis>-mediated starvation response in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.     

Heterogeneity in Colorectal Primary Tumor and Synchronous Liver Metastases

The expression profile of the ZEB1, ZEB2, VIM, CDH1, SFRP2, FOXQ1, TNC, MACC1, PLS3, CFTR, FLNA, MUC2, TFF3, and RARRES3 genes, as well as the mutational status of the KRAS, NRAS, BRAF, and PIK3CA genes, were investigated in 40 patients with colorectal cancer and liver metastases. A comparative analysis of changes in gene expression in primary tumor cells and liver metastases was performed. Statistically significant differences were found between the expression levels of the ZEB2 (p = 0.004), VIM (p < 0.001), FLNA (p = 0.04), and MUC2 (p < 0.001) genes. It was demonstrated that the overall frequency of mutations of the KRAS gene was 18/40 (45%) and the PIK3CA gene was 9/40 (23%). Mutations in the NRAS and BRAF genes were not found. The concordance between the primary tumor and metastases in the liver by mutation status was 100%.     

Pancreatic lipase inhibitory activity of monogalactosyldiacylglycerols isolated from the freshwater microalga <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Chemical investigation of the freshwater microalga Chlorella sorokiniana led to the isolation of a monogalactosyldiacylglycerol (MGDG)-rich fraction possessing dose-dependent inhibitory activity against pancreatic lipase activity. The MGDG-rich fraction contains 12 MGDGs identified by LC/HRMS analysis. Among them, three MGDGs were new compounds, namely, (2S)-1-O-(7Z,10Z-hexadecadienoyl)-2-O-(7Z,10Z,13Z-hexadecatrienoyl)-3-O-β-D-galactopyranosylglycerol (1), (2S)-1-O-linoleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (6), and (2S)-1-O-oleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (8). The major galactolipids were isolated by semipreparative HPLC and tested for their effect toward pancreatic lipase inhibitory activity. All the tested MGDGs showed significant reduction of pancreatic lipase activity indicating possible beneficial use for management of lipase-related disorders such as obesity.     

Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> cellobiohydrolase I expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To modify two main N-glycosylation residues of cellobiohydrolase I from Trichoderma reesei by site-directed mutagenesis for decreasing the extent of glycosylation and exploring possible effects on its properties.

Results

Asparagine 45 and 64 residues were mutated to alanine to make single/double mutants and expressed in P. pastoris. Decreasing N-glycosylation of the recombinant CBH I resulted in an increased affinity of the enzyme for carboxymethylcellulose and also improved the Kcat/Km while the specific activity was decreased. Also, the enzymes were stable up to 80 °C. There was no significant change of the optimum pH and temperature by decrease of glycosylation in the mutated enzymes in comparison to the wild-type at constant incubation time of assay.

Conclusion

Post-translational glycan-modification of CBH I in P. pastoris has different impacts on the properties of the secreted enzymes. Substrate affinity and catalytic efficiency were improved significantly while the activity and high temperature stability were negatively affected.

     

Dependence of expression of regulatory master genes of embryonic development in pancreatic cancer cells on the intracellular concentration of the master regulator PDX1

Exogenous expression of the gene encoding the pancreatic master regulator PDX1 in cell lines with different degrees of differentiation of pancreatic cancer cells is accompanied by changes in the expression of known master genes involved in cancer progression. In BxPC3^PDX+ cells, as compared to BxPC3^PDX–, we detected an increased expression of the following genes: NKX6.1 (2 times), NR5A2 (2.5 times), KLF5 (1.8 times), ZEB1 (3 times), and ONECUT1 (1.3 times), as well as a decreased expression of MUC1 and SLUG genes (3 and 2 times, respectively). In PANC1^PDX+ cells, as compared to the control PANC1^PDX– cells, we detected a decreased expression of ISL1 (2 times) and an increased expressed of KRT8 (2 times) and MUC1 (by 30%). In the high-grade cell lines (including the BxPC3 line studied), the total content of sites containing the marks of active enhancers was higher than that in the low-grade cell lines (PANC1).     

Analysis of the <Emphasis Type="Italic">Alu</Emphasis>I polymorphism in intron 1 of the human coagulation factor VIII gene: A new marker for the hemophilia a carrier detection

Frequencies of the C/T SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu?), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (C/T SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281–2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.     

Association analysis of alcohol metabolizing enzymes <Emphasis Type="Italic">ADH1B,ADH7, CYP2E1</Emphasis> gene polymorphism with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5′-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B ^* G (+MslI) allele, CYP2E1 ^* C2 (+PstI) allele and CYP2E1 ^* C (-DraI) allele were 8.48 ± 1.86, 1.20 ± 0.69, and 10.00 ± 1.90%, respectively. The ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7 ^* C (-StyI) allele was 44.58 ± 3.21%. A significantly higher frequency of CYP2E1 PstI C2 allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03–20.01). The tendency to significant effect of A1A2 genotype in ADH1B MslI polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0.042) were also revealed. Association of A1A2 genotype in ADH1B MslI polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.