Various oxidases and hydrolytic enzymes were analyzed to investigate the relationship between these enzymes and the skin pathogenicity of 18 Mucorales strains. Each strain was cultured in a nutrient medium containing starch as a carbon source. The cells grew quickly and were at a good state of growth after incubation for three days. Oxidase activity was not detected in any strain, whereas Mucor spp. including Mucor racemosus IFM47053 typically had high alcohol dehydrogenase (ADH) activity and all the strains had catalase activity. The culture filtrate and the cell free extract of each strain were applied to APIZYM test system, which revealed that all the strains examined produced many hydrolytic enzymes both inside and outside their mycelia. In the case of Absidia corymbifera strains, lipase activity was comparatively high, and polysaccharide hydrolytic enzymes such as alpha-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and alpha-fucosidase were produced.
Absidia ; Alcohol Dehydrogenase ; alpha-Glucosidases ; alpha-L-Fucosidase ; alpha-Mannosidase ; beta-Glucosidase ; Carbon ; Catalase ; Hydrolases ; Lipase ; Mucor ; Mucorales* ; Oxidoreductases ; Skin ; Starch ; Virulence ; Zygomycosis*

Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta- N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80 % of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba extracts than in culture media up to 120 hours after cultivation, but after 120 hours of cultivation those activities were detected higher in culture media than in the amoeba lysates. Neutral proteinase activity in A. culbertsoni increased more in EBSS medium than in the lysate specimens although the activity in the extracts was generally steady according to the cultivation time. Summarizing the above results, it is concluded that there were differences in hydrolase activities between pathogenic A. culbertsoni and non-pathogenic A. royreba, and that some hydrolase activities were detected remarkably higher in A. culbertsoni which revealed strong cytotoxicity to the target CHO cell line.
parasitology-protozoa ; Acanthamoeba culbertsoni ; Acanthamoeba royreba ; biochemistry ; hydrolase ; acid phosphatase ; beta-N-acetyl galactosaminidase ; beta-N-acetyl glucosaminidase ; alpha-mannosidase ; neutral proteinase ; acid proteinase ; mouse ; hydrolase ; acid phosphatase ; beta-N-acetyl galactosaminidase ; beta-N-acetyl glucosaminidase ; alpha-mannosidase ; neutral proteinase ; acid proteinase

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.
Bacillus subtilis ; enzymology ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Activation ; drug effects ; Hydrogen-Ion Concentration ; Kinetics ; Mannosidases ; chemistry ; isolation & purification ; metabolism ; Mercury ; pharmacology

Lysosomal alpha-mannosidase (EC 3.2.1.24) is a major exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease, alpha-mannosidosis, which has been described in humans, cattle, domestic cats and guinea pigs. Recently, great progress has been made in studying the enzyme and its deficiency. This includes cloning of the gene encoding the enzyme, characterization of mutations related to the disease, establishment of valuable animal models, and encouraging results from bone marrow transplantation experiments.
Animal ; Cats ; Cattle ; Cloning, Molecular ; Disease Models, Animal ; Guinea Pigs ; Human ; Lysosomes/*enzymology ; Mannosidases/*deficiency/*genetics/metabolism ; Mannosidosis/diagnosis/*etiology/*therapy ; Mutation ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Transcription, Genetic

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway and is encoded by a 3.0 kb cDNA. A 2.3 kb cDNA from a minor species of HeLa cell mRNA was discovered by RT-PCR cloning. Southern blotting and PCR analysis of the HeLa cell genomic DNA showed that the 2.3 kb message was encoded by the lysosomal alpha-mannosidase gene. Sequence comparison of the cDNA with the corresponding genomic DNA indicated that the 2.3 kb message was generated by an unusual intra-exonic joining event.
*Alternative Splicing ; Base Sequence ; DNA, Complementary/genetics ; Exons ; Hela Cells ; Human ; Lysosomes/*enzymology ; Mannosidases/*genetics ; Molecular Sequence Data ; RNA, Messenger/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Support, U.S. Gov't, P.H.S.

OBJECTIVETo study the effect of human alpha-mannosidase Man2c1 transgene on tumor growth and metastasis in mice.

METHODSHepatoma cell H22 or squamous epithelial carcinoma cell S180 was subcutaneously inoculated into the right armpit of mice (wild type mice and 28#, 35#, and 54# transgenic mice). Tumor size was measured every week. Mice were sacrificed on day 9 or 10 and then the tumors were exercised and weighted. Tumors and lungs were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The red blood cells in spleen were destroyed by Tris-NH4Cl. Natural killer (NK) cell activity was detected with Yac-1 cell as target.

RESULTSH22 and S180 tumors grew faster in all the three transgenic mice (28#, 35#, and 54#) than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P<0.05). Most tumors in the transgenic mice invaded the surrounding tissues. In contrast, nearly all the tumors in wild type mice were capsulized. Three of 10 28# transgenic mice, 5 of 10 35# transgenic mice, 3 of 10 54# transgenic mice, and 1 of 10 wild type mice showed lung metastasis of H22 tumor. Two of 6 28# transgenic mice, 3 of 6 35# transgenic mice, 1 of 6 54# transgenic mice, and 0 of 6 wild type mice showed lung metastasis of S180 tumor. No difference of NK activity in spleen cells was observed between the transgenic mice and wild type mice.

CONCLUSIONShMan2c1 transgene promotes growth, invasion, and metastasis of transplanted H22 and S180 tumors in mice. hMan2cl transgene does not affect NK activity in splenocytes.

Animals ; Cell Line, Tumor ; Humans ; Killer Cells, Natural ; immunology ; Lung Neoplasms ; secondary ; Mannosidases ; genetics ; Mice ; Mice, Transgenic ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Neoplasms, Experimental ; immunology ; metabolism ; pathology ; Spleen ; immunology ; Transgenes

Aims: This present study focused on purification of fungal &beta;-mannanase produced by Aspergillus niger USM F4 and also physicochemical characterisation of the purified enzyme. Methodology and results: The purified &beta;-mannanase with a molecular mass of ~47.4 kDa was demonstrated on SDSPAGE gel. The enzyme signified a purification degree of 4-fold, with final specific activity of 196.42 U/mg. It reached an optimum catalytic activity at pH 4.0 and 60 °C. The thermal stability of the enzyme was up to 70 °C and maintained the 50% activity after 30 min at 80 °C. Meanwhile, the pH stability was in the range of pH 3.0-9.0 and a 30 min half-life at pH 10.0. All chemical substances manifested an inhibitory effect on purified &beta;-mannanase, with SDS (28.16 ± 0.05% residual activity) as the strongest inhibitor, followed by cupric ion (Cu2+) (49.51 ± 0.09% residual activity). As a whole, the enzyme displayed a substrate specificity in the order of locust bean gum (LBG) > carboxymethylcellulose > soluble starch > xylan from oat spelt > &alpha;-cellulose. Its preference for LBG has generated the Km and Vmax values of 0.20 mg/mL and 9.82 U/mL, respectively. Conclusion, significance and impact of study The outcomes of our study offer potential for use at industrial scales, particularly in the oligosaccharides production that involve acid-related activity, wide-ranging temperature and pH stability.
Aspergillus niger ; beta-Mannosidase

OBJECTIVETo define the differences in gene expression patterns between glycidyl methacrylate (GMA)-transformed human lung fibroblast cells (2BS cells) and controls.

METHODSThe mRNA differential display polymerase chain reaction (DD-PCR) technique was used. cDNAs were synthesized by reverse transcription and amplified by PCR using 30 primer combinations. After being screened by dot blot analysis, differentially expressed cDNAs were cloned, sequenced and confirmed by Northern blot analysis.

RESULTSEighteen differentially expressed cDNAs were cloned and sequenced, of which 17 were highly homologous to known genes (homology = 89%-100%) and one was an unknown gene. Northern blot analysis confirmed that eight genes encoding human zinc finger protein 217 (ZNF217), mixed-lineage kinase 3 (MLK-3), ribosomal protein (RP) L15, RPL41, RPS 16, TBX3, stanniocalcin 2 (STC2) and mouse ubiquitin conjugating enzyme (UBC), respectively, were up-regulated, and three genes including human transforming growth factor beta inducible gene (Betaig-h3), alpha-1,2-mannosidase 1A2 (MAN 1A2) gene and an unknown gene were down-regulated in the GMA-transformed cells.

CONCLUSIONAnalysis of the potential function of these genes suggest that they may be possibly linked to a variety of cellular processes such as transcription, signal transduction, protein synthesis and growth, and that their differential expression could contribute to the GMA-induced neoplastic transformation.

Air Pollutants, Occupational ; toxicity ; Carcinoma, Squamous Cell ; genetics ; pathology ; Cell Line, Transformed ; Epoxy Compounds ; toxicity ; Fibroblasts ; cytology ; drug effects ; Gene Expression Profiling ; Glycoproteins ; metabolism ; Humans ; Lung ; cytology ; Male ; Mannosidases ; drug effects ; metabolism ; Methacrylates ; toxicity ; Mitogen-Activated Protein Kinase 3 ; drug effects ; metabolism ; Oligonucleotide Array Sequence Analysis ; Ribosomal Proteins ; metabolism ; Signal Transduction ; genetics ; Transforming Growth Factor beta ; drug effects ; metabolism ; Ubiquitins ; metabolism ; Zinc Fingers ; drug effects ; physiology

OBJECTIVETo study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB-F344 and the growth of the cells in culture.

METHODSRecombinant adeno-associated virus (rAAV) expression vector pAGX (+) containing an antisense or a sense fragment of 6A8 cDNA encoding a human alpha-mannosidase was constructed. The recombinant vectors or the mock were transfected into WB-F344 cells by means of lipofectAmine. The transfected cells were selected in G418 medium and cloned by means of limiting dilution. Integration of the transfected DNA into host DNA was detected by neo PCR. Rat liver ER alpha-mannosidase activity in cell supernatant was measured by using P-nitrophenyl-alpha-D-mannopyranoside as a substrate. Microvilli on cell surface were observed upon a scan electron microscope. The growth curves of the cells in culture were drawn.

RESULTSThe cell clones transfected with antisense 6A8 showed reduction of ER alpha-mannosidase activity with various degrees. Clone AS1 and AS2 cell showed a pronounced reduction of the enzymatic activity. In the study on AS1 cells, Con A binding to the cells was found to be enhanced, cell growth in culture became slow from day 5. The microvilli on the cells were reduced and blunted.

CONCLUSIONSTransfection with antisense 6A8 resulted in reduction and blunting of microvilli on the surface of growing WB-F344 cells, which might be related to N-glycosylation modification.

Animals ; Cloning, Molecular ; Epithelial Cells ; cytology ; Glycosylation ; Liver ; cytology ; Microvilli ; Rats ; Transfection ; alpha-Mannosidase ; genetics ; metabolism

OBJECTIVETo detect the differential display of mRNA expression between human nasopharyngeal carcinoma cell CNE-2L2 with reduced malignancy caused by transduction of a DNA antisense to 6A8 alpha-mannosidase cDNA (AS cell) and the wild type cell (W cell).

METHODSDifferential display of mRNA expression was analyzed using DNA microarray analysis. The datasets were confirmed by Northern blotting and RT-PCR.

RESULTSOut of the 1069 genes analyzed, 34 genes were up-regulated in AS cells relative to W cells. Conversely, 42 genes were down-regulated. The genes, up-regulation of which might have suppressive effect on tumor malignant behaviors, were P130 mRNA for 130K protein, TGF-betaIIR alpha, GABBR1, TGFBR1, TNFAIP1, STANIN, E-CADHERIN, CTNNA1 and 2, RFX2, TMPO, etc. The genes, down-regulation of which might have suppressive effect on tumor malignant behaviors, were CD44, NDRG1, TGFB1, RPS5, LEGUMAIIN, CBS, CD59, SNRPA1, etc. The microarray datasets were confirmed by Northern blot and RT-PCR analysis.

CONCLUSIONSIn comparison to the W cell, AS cell has up-regulation of 34 genes and down-regulation of 42 genes. Changes of the gene expression may play a role in the malignancy reduction of AS cell.

Gene Expression Profiling ; Humans ; Nasopharyngeal Neoplasms ; enzymology ; genetics ; Oligonucleotide Array Sequence Analysis ; Tumor Cells, Cultured ; alpha-Mannosidase ; biosynthesis ; genetics