Alkaline phosphatase isozymes as possible markers of differentiation in human testicular teratocarcinoma cell lines

The alkaline phosphatase (ALP) activity in eight independent cell lines derived from human testicular germ cell tumors was characterized. Seven out of eight of the lines had high ALP levels, and most of the activity in each case was of the liver/bone/kidney ALP type, as judged by thermostability, inhibition, and electrophoretic studies. Low levels of a heat stable, placental-like ALP were also present in these seven lines; only a small subpopulation of the cells of each line reacted strongly with an anti-placental ALP monoclonal antibody. The heat-stable, placental-like isozyme characteristic of these lines differed from the normal placental ALP in its inhibition profile. Thus it is possible that a subpopulation of the cells in these lines expresses a new embryonic ALP form.     

Alkaline phosphatase expression in human cell lines derived from primary hepatomas

Isozyme patterns of alkaline phosphatase (ALP) were electrophoretically examined in human cell lines derived from one hepatoblastoma, five hepatocellular carcinomas (HCCs) and two cholangiocellular carcinomas. Most of the cell lines tested had a liver-type ALP isozyme. In addition, an abnormal ALP isozyme, which was similar to variant ALP, was detected in one hepatoblastoma and two HCC cell lines. One HCC cell line of these variant-like ALP-positive cell lines was alpha-fetoprotein (AFP)-negative. These findings suggest that variant-like ALP may be useful for the identification of human hepatoma cell lines, especially in AFP or albumin-negative cell lines.     

Characterization and expression of uterine and placental alkaline phosphatases in the mouse.

Alkaline phosphatase (ALP) is rapidly induced in the uterine subepithelial stroma after a natural or artificial decidual stimulus. During gestation ALP-specific activity peaked at Day 7 to 8 (Day 1 is day of detection of the copulation plug) followed by a rapid decline to control levels by Day 9. This elevation in enzyme activity was preceded by an 8-fold induction of a 2.6 kilobase (kb) mRNA. This mRNA was not preferentially localized to implantation sites. ALP activity was detected in the placenta at Day 9 and reached maximum specific activity at Day 19. The placental ALP was also encoded by a 2.6 kb mRNA. Uterine and placental ALPs were inhibited to the same extent by levamisole, L-tryptophan and homoarginine. The calculated Ki values for these inhibitors were not statistically different between the uterine and placental forms. Km values towards the substrate p-nitrophenylphosphate, however, were statistically different between the uterine and placental forms. Both uterine and placental ALPs were stimulated 3-4-fold by addition of 2 mM-Mg2+. Electrophoretic mobilities on SDS polyacrylamide gel, where the enzyme migrated as a single band, were the same. The uterine form, however, could be distinguished from the placental isoenzyme by separation on non-denaturing polyacrylamide gels; the uterine form had a single zone of activity which migrated with an intermediate mobility between the two zones of activity detected for the placental enzyme. These differences in mobility could be ascribed to the sialic acid content of the enzyme because treatment with neuraminidase resulted in the uterine and placental forms migrating with comparable but slower mobilities in non-denaturing gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation and quantification of serum alkaline phosphatase isozymes in the rat by affinity electrophoresis

The purpose of this study was to examine the possibility of separation and quantification of serum alkaline phosphatase (ALP) isozymes in rats by wheatgerm lectin affinity electrophoresis. Cellulose acetate electrophoresis of the liver and bone ALPs without lectin results in overlapping bands, but in the presence of lectin, the mobility of the band of bone enzyme was retarded and well separated from the liver enzyme band. With this affinity electrophoretic method, we determined the serum ALP isozymes in fed and fasting rats grouped by age. As a result, the absolute activity of bone isozyme showed a downward trend with age in the fed and fasting rats. The serum ALP activity was steadily higher in fed rats than in fasting rats, and the increase was due to intestinal ALP isozyme. There was low activity bordering complete absence in liver isozyme under both nutritional conditions. The affinity electrophoretic method provided a rapid, reproducible, and relatively simple technique for further clinical characterization of ALP isozyme in the rat serum.     

Use of monoclonal antibodies to assign phenotypes to placental alkaline phosphatase from cultured human cell lines derived from tumors

Monoclonal antibodies were used to type placental alkaline phosphatase (ALP) from cell lines established from malignant human tumors by incubating ALP extracts from the cells with antibodies of different allelic specificities and separating free from bound enzyme on polyacrylamide gel electrophoresis. The HeLa-derived cell lines (Hep 2 and WISH) have the type 1 ALP phenotype, while a non-HeLa cell line (HT-3) has the type 2 ALP phenotype. This approach should prove of value for the phenotyping of enzymes and proteins with poorly resolved or altered electrophoretic patterns.     

通过“供体-受体”原生质体融合将裸燕麦部分核基因组转移给普通小麦(英文)

本文进行了小麦和裸燕麦悬浮细胞原生质体的电融合,并基于双亲失活(用IOA处理受体小麦原生质体,用γ-射线照射供体裸燕麦细胞系),获得可能的杂种愈伤组织。对7块愈伤组织进行了乙醇脱氢酶(Adh)同工酶筛选,发现5块表现出双亲特征酶带。对3个杂种细胞系进行5种同工酶分析,证实它们均为稳定的不对称体细胞核杂种细胞系;它们表现出小麦的完整谱带和裸燕麦的部分谱带。对2个杂种细胞系及亲本的核糖体DNA Southern分析结果表明只有一个杂种细胞系(HB 95)含有双亲的全部谱带。细胞学观察表明,2个杂种细胞系的染色体数目均显著高于双亲。从杂种细胞系HB 94中分化出叶原基等分化结构。Adh同工酶分析表明,这些分化结构具有和母体细胞系完全相同的杂种谱带。     

Identification of a novel aspartic-like protease differentially expressed in human breast cancer cell lines

Four different human breast cancer cell lines were examined to search for genes associated with tumor growth and metastasis. Each of these cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and MDA-MB-435, displays different phenotypic characteristics ranging from poorly to highly tumorigenic and metastatic. The differences in gene expression profiles of these cell lines generated by differential display technique should allow one to identify candidates as putative oncogenes or tumor/metastasis suppressor genes. A novel cDNA expressed in the highly tumorigenic and metastatic cell line, MDA-MB-435, was identified and isolated by this approach. The function for this gene, designated ALP56 (aspartic-like protease 56 kDa), in tumor progression is suggested by the homology of the encoded protein to aspartic proteases, such as cathepsin D. The amino acid residues in two catalytic domains of this family are highly conserved in those domains of ALP56. Northern hybridization indicated that the expression of ALP56 is associated with growth and metastasis of MDA-MB-435 tumors in immunodeficient mice. In situ hybridization of biopsies from breast cancer and colon cancer patients indicated that ALP56 is upregulated in human primary tumors and liver metastasis. These results suggest that this novel gene correlates with human tumor progression.     

Genetic Transformation of Drosophila melanogaster with an Autonomous P Element: Phenotypic and Molecular Analyses of Long-Established Transformed Lines

Following transformation of a Drosophila melanogaster true M strain with an autonomous P element, six lines were established and monitored for their molecular and phenotypic properties during a 4-yr period. The number of P elements increased with time in all the lines but the rate of increase differed among lines. Furthermore, degenerate elements arose in each of the lines during propagation. By the end of the 4th yr, the total number of elements in every line was similar to that of a very strong P strain.--At the phenotypic level, all of the transformed lines evolved high P activity, but only three developed complete or nearly complete regulatory ability. The other three lines attained only intermediate levels of regulation over the 4-yr period. One of these lines was particularly noteworthy. Although it contained as many as 55 P elements per genome (20 of which were potentially complete) and had extremely high P activity potential, it continued to exhibit limited regulatory ability. In addition, when females of this line were maintained at high temperatures, the ability to suppress P activity was even further diminished. A strain with this combination of molecular and phenotypic properties, in an apparently stable configuration, has not been previously described.--The results are discussed in the context of the possible role of degenerate elements in regulating P element expression.     

Mathematical deconvolution uncovers the genetic regulatory signal of cancer cellular heterogeneity on resistance to paclitaxel

Drug resistance remains a major problem in combating malignancies, resulting critical the resistance to paclitaxel used in the treatment of many different cancers. Elucidating the cellular heterogeneity composition of tumours may be relevant to designing more effective treatment strategies on drug resistance. In particular, such heterogeneity correlates with the measurement of gene expression below the population level. However, experimental assays capturing differential response are limited and cannot discern the variation in gene expression specific to different cellular types in tumour populations. These limitations led us to consider a mathematical modelling approach, in which the gene expression of cellular subpopulations is recovered by deconvolution. Mathematically, the deconvolution is a multi-linear regression-based problem. We combined herein data on cellular subpopulation frequency composition with gene expression values from 16 tumour lines (8 resistant and 8 sensitive to paclitaxel treatment) to find genes that are differentially expressed between paclitaxel resistant and paclitaxel sensitive tumour lines in different cellular subpopulations. The results indicate that many genes differentially expressed between paclitaxel resistant and sensitive cancer lines are only detected when considering their heterogeneous cellular composition. Overall, our methodology is thought to keep in mind phenotypic heterogeneity improving our resolution in the identification of biomarkers on resistance to chemo-therapeutic agents.     

Phenotypic and functional characterization of human thymic stromal cell lines.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.     

Introgression of the heterologous nuclear DNAs and efficacious compositions from Swertia tetraptera Maxim. into Bupleurum scorzonerifolium Willd. via somatic hybridization

Swertia tetraptera Maxim. is an important source of secoiridoid glucosides. To produce these pharmacologically valuable compounds heterologously in somatic hybrid cell lines, S. tetraptera protoplasts were irradiated with various doses of UV light and fused with protoplasts from a long-term cell line of Bupleurum scorzonerifolium Willd. This recipient was chosen as the cell line is cytogenetically stable and fast growing; furthermore, protoplasts isolated from the cell line are readily regenerable. From a set of 86 putative hybrid calli, only two were able to regenerate viable green plants. The hybridity of the 19 of the 86 selections was revealed by a combined isozyme and RAPD analysis, supported by a karyotypic study based on genomic in situ hybridization (GISH). Clone I-3 contained 0.014% swertiamarin while the regenerants had 0.069% swertiamarin and 0.409% gentiopicroside while the III-4 plants contained only 0.015% gentiopicroside.     

Sperm mobility: phenotype in roosters (Gallus domesticus) determined by mitochondrial function

Previously, inheritance of sperm mobility entailed a maternal additive genetic effect, and sperm ATP content was correlated (r = 0.80) with phenotype. The present study was conducted to determine if mitochondrial function was critical to phenotypic expression. Whereas phenotype was independent of mitochondrial helix length, phenotype was correlated with sperm oxygen consumption (r = 0.83) using random-bred roosters. Aberrant mitochondria characterized immobile sperm, as evidenced by transmission-electron microscopy. Such mitochondria were swollen and contained disorganized cristae. Additional experiments were performed with roosters from lines selected for low or high sperm mobility. A threefold difference in sperm oxygen consumption was observed between lines. Single nucleotide polymorphisms were observed in mitochondrial DNA by sequencing replicate mitochondrial genomes from each line. An A-to-G substitution in the gene encoding tRNA(Arg) was inherited consistently, as evidenced by restriction fragment length polymorphism analysis using two male and two female progeny per family group and 14 family groups per line. Motile concentration in semen from low-line males was half that observed in semen from high-line males, as evidenced by computer-assisted sperm motion analysis. Likewise, 47% of sperm from low-line males contained aberrant mitochondria, compared to 4% for high-line males. In summary, sperm mobility phenotype was dependent on mitochondrial function, which in turn was altered by genetic selection. Fowl deferent duct fluid contains a high concentration of glutamate. We propose that variation in sperm mobility phenotype stems from the extent to which glutamate induces excessive mitochondrial Ca2+ uptake before ejaculation.     

Activity of gut alkaline phosphatase,proteases and esterase in relation to diapause of pharate first instar larvae of the gypsy moth,Lymantria dispar

Two distinctly different patterns of gut enzyme activity were noted in relation to diapause in pharate first instar larvae of the gypsy moth, Lymantria dispar. Trypsin, chymotrypsin, elastase, aminopeptidase and esterase activities were low at the initiation of diapause and through the period of chilling needed to terminate diapause. At the completion of a 150 day chilling period, activity of each of these enzymes quickly increased when the pharate larvae were transferred to 25°C. By contrast, activity of alkaline phosphatase (ALP) increased rapidly at the onset of diapause, remained elevated throughout diapause, increased again during postdiapause, and then dropped at the time of hatching. In addition, zymogram patterns of ALP activity differed qualitatively in relation to diapause: several bands were detectable during the pre- and postdiapause periods, but only one band, a band of high mobility, was visible during diapause. The ALP isozyme present in diapausing pharate larvae had a pH optimum of 10.6. Diapause in the gypsy moth can be averted by application of an imidazole derivative, KK-42, and pharate larvae treated with KK-42 showed elevated protease and esterase activity, low ALP activity, and expressed ALP isozymes with low mobility. Thus the overall patterns of gut enzyme activity and the ALP zymogram in KK-42 treated individuals were similar to those observed in untreated individuals at the termination of diapause. Our results suggest a unique pattern of enzyme activity in the gut that is regulated by the diapause program. Arch. Insect Biochem. Physiol. 37:197–205, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.     

Isolation and characteristics of autoreactive T cells specific to aggrecan G1 domain from rheumatoid arthritis patients

NTRODUCTIONRheumatoid arthritis (RA) is a common disease characterized by the chroniclesion of polyarthritis. The etiology and pathogenesis of RA remain unknown. Autoimmunity to cartilage alltigens may play a significant role in the pathogenesis ofchronic inflammatory polyarthritis. It is commonly accepted that cell mediated immune responses are involved in chronic inflammation since T and B lymphocytes andatigen presenting cells were observed to be enriched in the synovium fluid of RA…     

Variation of Storage Proteins and Isozymes within Maize Inbred Lines

Seed storage proteins of ten maize inbred lines were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, fourteen isozyme loci representing nine isozyme systems were analysed. Salt-soluble protein fraction contained a large number of proteins (30 – 40 bands) of different sizes with genotypic differences among the ten inbred lines. The methionine-rich 10 kD zein showed differential expression in the ten inbred lines with different migration rates on the SDS-PAGE. This polypeptide was completely absent in the inbred line G221D. Among nine of the inbred lines, eight of 14 isozyme loci were polymorphic and six were monomorphic resulting in seven unique fingerprints.     

Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells.

Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.     

Identification of differentially expressed genes in isogenic highly metastatic and poorly metastatic cell lines of R3230AC rat mammary adenocarcinoma

Tumour metastasis occurs as a result of a cascade of events including alterations in the expression of various genes. The identification of such genes is essential to understanding formation of metastasis. In a previous study, highly metastatic (LN4.D6) and poorly metastatic (CAb.D5) cell lines were obtained from the rat mammary adenocarcinoma cell line R3230AC. Subtractive hybridization was used to identify differentially expressed genes between these two cell lines. We identified eight cDNA clones in CAb.D5 and six cDNA clones in LN4.D6 that were differentially expressed. One of the cDNA clones in each cell line had no homology with known sequences. Expression patterns of these differentially expressed genes were examined in a pair of rat mammary and prostate adenocarcinoma cell lines. Compared with cell lines examined, cDNA FF-10 was only expressed in CAb.D5; however, cDNA RB-8, RE-1, RF-5 were only expressed in the highly metastatic LN4.D6. No correlation was observed between expression patterns of the differentially expressed genes and metastatic potential of these cells. However, differential expression of genes, especially cytokeratins (CK8 and CK5) and collagens (III and IV) between highly metastatic and low metastatic rat mammary adenocarcinoma cell lines might initiate further investigation of these genes in metastatic process.     

Secretion of antileucoprotease from a human lung tumor cell line

Two human tumor cell lines were analyzed for the production of human antileucoprotease (ALP). One of them, a human squamous lung carcinoma cell line (HS-24) synthesized, as confirmed by Western blot analysis, high amounts of ALP in serum-free medium. The supernatant inhibited elastase, chymotrypsin and trypsin. Northern blot analysis with an 18-mer radiolabelled oligonucleotide, derived from an ALP specific cDNA clone, revealed a specific mRNA of about 700-800 nucleotides in HS-24 tumor cells. In contrast, a secondary human lung tumor cell line (SB-3), derived from the adrenal cortex, did not synthesize ALP when assayed under identical conditions. The supernatant inhibited only trypsin and chymotrypsin.