The multiple forms and kinetic properties of the N-acetyl-beta-D-hexosaminidases from colonic tumours and mucosa of rats treated with 1,2-dimethylhydrazine.
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ABSTRACT:
SUBMITTER: Mian N
PROVIDER: S-EPMC1186371 | biostudies-other | 1979 Jan
REPOSITORIES: biostudies-other
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Project description:BackgroundAzoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats.MethodsFor gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 x 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 x 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent).ResultsMicroarray gene expression analysis showed that Defcr4, Igfbp5, Mmp7, Nos2, S100A8 and S100A9 were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while Slc26a3, Mptx, Retlna and Muc2 were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFalpha/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including Apc.ConclusionThe results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to note that one of the alterations concerned Apc, a key gene in colorectal carcinogenesis. The fact that many of the molecular alterations described in this study are documented in human colon tumours confirms the relevance of DMH-induced cancers as a powerful tool for the study of colon carcinogenesis and chemoprevention.
| S-EPMC2877689 | biostudies-literature
Project description:BackgroundChronic inflammation is well known to be a risk factor for colon cancer. Previously we established a novel mouse model of inflammation-related colon carcinogenesis, which is useful to examine the involvement of inflammation in colon carcinogenesis. To shed light on the alterations in global gene expression in the background of inflammation-related colon cancer and gain further insights into the molecular mechanisms underlying inflammation-related colon carcinogenesis, we conducted a comprehensive DNA microarray analysis using our model.MethodsMale ICR mice were given a single ip injection of azoxymethane (AOM, 10 mg/kg body weight), followed by the addition of 2% (w/v) dextran sodium sulfate (DSS) to their drinking water for 7 days, starting 1 week after the AOM injection. We performed DNA microarray analysis (Affymetrix GeneChip) on non-tumorous mucosa obtained from mice that received AOM/DSS, AOM alone, and DSS alone, and untreated mice at wks 5 and 10.ResultsMarkedly up-regulated genes in the colonic mucosa given AOM/DSS at wk 5 or 10 included Wnt inhibitory factor 1 (Wif1, 48.5-fold increase at wk 5 and 5.7-fold increase at wk 10) and plasminogen activator, tissue (Plat, 48.5-fold increase at wk 5), myelocytomatosis oncogene (Myc, 3.0-fold increase at wk 5), and phospholipase A2, group IIA (platelets, synovial fluid) (Plscr2, 8.0-fold increase at wk 10). The notable down-regulated genes in the colonic mucosa of mice treated with AOM/DSS were the peroxisome proliferator activated receptor binding protein (Pparbp, 0.06-fold decrease at wk 10) and the transforming growth factor, beta 3 (Tgfb3, 0.14-fold decrease at wk 10). The inflammation-related gene, peroxisome proliferator activated receptor gamma (Ppargamma 0.38-fold decrease at wk 5), was also down-regulated in the colonic mucosa of mice that received AOM/DSS.ConclusionThis is the first report describing global gene expression analysis of an AOM/DSS-induced mouse colon carcinogenesis model, and our findings provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies against carcinogenesis.
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Project description:Fermentation of dietary fiber in the colon results in the production of short chain fatty acids (mainly propionate, butyrate and acetate). Butyrate modulates a wide range of processes, but its mechanism of action is mostly unknown. This study aimed to determine the effects of butyrate on the transcriptional regulation of human colonic mucosa in vivo.Five hundred genes were found to be differentially expressed after a two week daily butyrate administration with enemas. Pathway analysis showed that the butyrate intervention mainly resulted in an increased transcriptional regulation of the pathways representing fatty acid oxidation, electron transport chain and oxidative stress. In addition, several genes associated with epithelial integrity and apoptosis, were found to be differentially expressed after the butyrate intervention.Colonic administration of butyrate in concentrations that can be achieved by consumption of a high-fiber diet enhances the maintenance of colonic homeostasis in healthy subjects, by regulating fatty acid metabolism, electron transport and oxidative stress pathways on the transcriptional level and provide for the first time, detailed molecular insight in the transcriptional response of gut mucosa to butyrate.
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Project description:The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.
| S-EPMC1158494 | biostudies-other
Project description:Human N-acetyl-beta-hexosaminidases As and P are probably sialylated since they are susceptible to neuraminidase attack. twhen infused into the circulation of a rat they are removed more slowly than the non-sialylated forms of the enzyme from tissue and urine.
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Project description:Colonoscopy biopsies from patients with ulcerative colitis were collected prior to biological therapy. The sampled mucosa was the area of active lesion. Heterogenety of the samples were studied.
2022-12-17 | GSE220815 | GEO
Project description:The mucosa-associated microbiota is an important component in human microbiota. The aim was to investigate mucosa-associated microbiota using brush samples during endoscopic procedures and compare with fecal microbiota. Seven patients who were planning to undergo a routine colonoscopy were recruited. Mucosal brushing samples were taken from 3 sites (terminal ileum, ascending and sigmoid colon), and a fecal sample was taken on the morning of colonoscopy. The samples were immediately placed in microcentrifuge tubes containing DNA stabilization reagent and analyzed using the next generation sequencer. The individual differences in microbiota were more evident than the differences of the sampling sites. Actinobacteria was more abundant and Bacteroidetes was less in the brush samples than those in the fecal samples. Taxonomic composition at the genus level and the proportion of genes responsible for some functions in the brushing samples tended to be different from those in the fecal samples. Bulleidia and Oribacteriumi were significantly more abundant and the proportions of genes responsible for transcription factors and phosphotransferase system were higher in ileal mucous than those in fecal samples. Brushing during colonoscopic procedure instead of using feces samples might be useful to analyze mucosa-associated microbiota.
| S-EPMC6769413 | biostudies-literature
Project description:Despite advances in perioperative care, the postoperative mortality rate after emergency oncological colonic resection remains high. Risk stratification may allow targeted perioperative optimization and cardiac risk stratification. This study aimed to test the hypothesis that the Revised Cardiac Risk Index (RCRI), a user-friendly tool, could identify patients who would benefit most from perioperative cardiac risk mitigation. Patients who underwent emergency resection for colonic cancer from 2007 to 2017 and registered in the Swedish Colorectal Cancer Registry (SCRCR) were analysed retrospectively. These patients were cross-referenced by social security number to the Swedish National Board of Health and Welfare data set, a government registry of mortality, and co-morbidity data. RCRI scores were calculated for each patient and correlated with 90-day postoperative mortality risk, using Poisson regression with robust error of variance. Some 5703 patients met the study inclusion criteria. A linear increase in crude 90-day postoperative mortality was detected with increasing RCRI score (37.3 versus 11.3 per cent for RCRI 4 or more versus RCRI 1; P < 0.001). The adjusted 90-day all-cause mortality risk was also significantly increased (RCRI 4 or more versus RCRI 1: adjusted incidence rate ratio 2.07, 95 per cent c.i. 1.49 to 2.89; P < 0.001). This study documented an association between increasing cardiac risk and 90-day postoperative mortality. Those undergoing emergency colorectal surgery for cancer with a raised RCRI score should be considered high-risk patients who would most likely benefit from enhanced postoperative monitoring and critical care expertise.
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Project description:We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilization of a putative noncanonical truncation pathway in favor of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate PMP formation via a putative noncanonical truncation pathway in neutrophils.
| S-EPMC8966476 | biostudies-literature
Project description:β-N-acetyl-D-hexosaminidases have attracted significant attention due to their crucial role in diverse physiological functions including antibacterial synergists, pathogen defense, virus infection, lysosomal storage, and protein glycosylation. In particular, the GH3 β-N-acetyl-D-hexosaminidase of V. cholerae (VcNagZ), human GH20 β-N-acetyl-D-hexosaminidase B (HsHexB), and human GH84 β-N-acetyl-D-hexosaminidase (hOGA) are three important representative glycosidases. These have been found to be implicated in β-lactam resistance (VcNagZ), lysosomal storage disorders (HsHexB) and Alzheimer's disease (hOGA). Considering the profound effects of these three enzymes, many small molecule inhibitors with good potency and selectivity have been reported to regulate the corresponding physiological functions. In this paper, the best-known inhibitors PUGNAc and two of its derivatives (N-valeryl-PUGNAc and EtBuPUG) were selected as model compounds and docked into the active pockets of VcNagZ, HsHexB, and hOGA, respectively. Subsequently, molecular dynamics simulations of the nine systems were performed to systematically compare their binding modes from active pocket architecture and individual interactions. Furthermore, the binding free energy and free energy decomposition are calculated using the MM/GBSA methods to predict the binding affinities of enzyme-inhibitor systems and to quantitatively analyze the contribution of each residue. The results show that PUGNAc is deeply-buried in the active pockets of all three enzymes, which indicates its potency (but not selectivity) against VcNagZ, HsHexB, and hOGA. However, EtBuPUG, bearing branched 2-isobutamido, adopted strained conformations and was only located in the active pocket of VcNagZ. It has completely moved out of the pocket of HsHexB and lacks interactions with HsHexB. This indicates why the selectivity of EtBuPUG to VcNagZ/HsHexB is the largest, reaching 968-fold. In addition, the contributions of the catalytic residue Asp253 (VcNagZ), Asp254 (VcNagZ), Asp175 (hOGA), and Asp354 (HsHexB) are important to distinguish the activity and selectivity of these inhibitors. The results of this study provide a helpful structural guideline to promote the development of novel and selective inhibitors against specific β-N-acetyl-D-hexosaminidases.
| S-EPMC6499197 | biostudies-literature