Embryonic alkaline phosphatase is expressed at M-phase in the spermatogenic lineage of the mouse

Development ◽  
1992 ◽  
Vol 116 (1) ◽  
pp. 159-165 ◽  
Author(s):  
S. Narisawa ◽  
M.C. Hofmann ◽  
C.A. Ziomek ◽  
J.L. Millan
Keyword(s):  
Alkaline Phosphatase ◽  
Present Report ◽  
Chinese Hamster ◽  
Adult Life ◽  
Ovary Cell ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Late Prophase ◽  
Newborn Mice ◽  
M Phase

We have recently cloned and characterized a novel embryonic alkaline phosphatase (EAP) expressed at the two-cell to blastocyst stage of preimplantation development in the mouse. The isozyme is re-expressed in trace amounts in the thymus, intestine and testis during adult life. In the present report, we find that EAP transcripts can be detected, by RT-PCR analysis, in very low amounts in the testes of newborn mice, but at 24 days of age EAP mRNA levels reach the highest concentrations, remaining high at 40 and even 117 days of age. We produced a synthetic peptide and a corresponding rabbit anti-peptide antiserum (Rb-1434), which was characterized by enzyme antigen immunoassays and reactivity with chinese hamster ovary cell transfectants, as reacting specifically with EAP. The Rb-1434 antibody enabled us to examine immunohistochemically what cell types in the testis are responsible for the expression of EAP during different developmental stages. No positive cells were recognized in the testis of newborns (day 0) and 8-day-old mice. Positive cells were first observed at day 15 and, at 24 days of age, many positive M-phase cells, morphologically corresponding to spermatocytes in mid to late prophase of meiotic division I, were strongly positive for EAP expression. Positive M-phase cells were also observed at 40 days and 151 days of age. Transgenic mice expressing the human GCAP isozyme in a tissue-specific manner in the testis, showed equivalent stages of M-phase figures when stained immunohistochemically with a specific rabbit polyclonal antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells

1985 ◽  
Vol 5 (12) ◽  
pp. 3525-3531
Author(s):  
J K Griffith
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Recombinant Dna ◽  
Chinese Hamster ◽  
Single Copy ◽  
Ovary Cell ◽  
Gene Copy ◽  
Mrna Levels ◽  
Protein Levels ◽  
Copy Numbers

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.

scholarly journals Characterization of β-adrenergic receptors in bovine intramuscular and subcutaneous adipose tissue: comparison of lubabegron fumarate with β-adrenergic receptor agonists and antagonists

2021 ◽  
Vol 99 (8) ◽  
Author(s):  
Jinhee H Hwang ◽  
Michael E Spurlock ◽  
John C Kube ◽  
Xiang Z Li ◽  
Stephen B Smith
Keyword(s):  
Adipose Tissue ◽  
Adrenergic Receptor ◽  
Quantitative Polymerase Chain Reaction ◽  
Animal Health ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Mrna Levels ◽  
Adipose Tissues

Abstract Chinese hamster ovary cell constructs expressing either the β 1-, β 2- or β 3-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β 3-AR subtype, with an EC50 of 6 × 10–9 M, with no detectible agonistic activity at the β 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β 1-, β2-, and β 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β 1-AA), salbutamol sulfate (SAL; specific β 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β 1-, β 2-, and β 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β 2-AR (P < 0.05), with the β 1- and β 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β 1- and β 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β 1- and β3-receptor subtypes. The minimal mRNA expression of the β 1- and β 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes.

scholarly journals Decreased catalytic subunit mRNA levels and altered catalytic subunit mRNA structure in a cAMP-resistant Chinese hamster ovary cell line

1991 ◽  
Vol 266 (16) ◽  
pp. 10189-10195
Author(s):  
P. Howard ◽  
K.H. Day ◽  
K.E. Kim ◽  
J. Richardson ◽  
J. Thomas ◽  
...  
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Catalytic Subunit ◽  
Ovary Cell ◽  
Mrna Levels ◽  
Subunit Mrna ◽  
Mrna Structure ◽  
Ovary Cell Line

scholarly journals Cloning ofTrypanosoma bruceiandLeishmania majorGenes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

2002 ◽  
Vol 277 (51) ◽  
pp. 50176-50182 ◽  
Author(s):  
Tunhan Chang ◽  
Kenneth G. Milne ◽  
Maria Lucia Sampaio Güther ◽  
Terry K. Smith ◽  
Michael A. J. Ferguson
Keyword(s):  
Cell Line ◽  
Leishmania Major ◽  
Essential Gene ◽  
Chinese Hamster ◽  
Sleeping Sickness ◽  
Ovary Cell ◽  
Mrna Levels ◽  
Null Mutant ◽  
African Sleeping Sickness ◽  
Inositol 1

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol (GlcNAc-PI) tod-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol by GlcNAc-PI de-N-acetylase. The genes encoding this activity arePIG-LandGPI12in mammals and yeast, respectively. Fragments of putative GlcNAc-PI de-N-acetylase genes fromTrypanosoma bruceiandLeishmania majorwere identified in the respective genome project data bases. The full-length genesTbGPI12andLmGPI12were subsequently cloned, sequenced, and shown to complement aPIG-L-deficient Chinese hamster ovary cell line and restore surface expression of GPI-anchored proteins. A tetracycline-inducible bloodstream formT. brucei TbGPI12conditional null mutant cell line was created and analyzed under nonpermissive conditions.TbGPI12mRNA levels were reduced to undetectable levels within 8 h of tetracycline removal, and the cells died after 3–4 days. This demonstrates thatTbGPI12is an essential gene for the tsetse-transmitted parasite that causes Nagana in cattle and African sleeping sickness in humans. It also validates GlcNAc-PI de-N-acetylase as a potential drug target against these diseases. Washed parasite membranes were prepared from the conditional null mutant parasites after 48 h without tetracycline. These membranes were shown to be greatly reduced in GlcNAc-PI de-N-acetylase activity, but they retained their ability to make GlcNAc-PI and to processd-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol to later glycosylphosphatidylinositol intermediates. These results suggest that the stabilities of other glycosylphosphatidylinositol pathway enzymes are not dependent on GlcNAc-PI de-N-acetylase levels.

TcNST2encodes a Golgi-localized UDP-galactose transporter inTrypanosoma cruzi

Parasitology ◽  
2019 ◽  
Vol 146 (11) ◽  
pp. 1379-1386 ◽  
Author(s):  
Elizabeth C. Rodrigues ◽  
Patricia Mörking ◽  
Jaqueline O. Rosa ◽  
Bruno A. A. Romagnoli ◽  
Beatriz G. Guimarães ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Three Dimensional ◽  
Chinese Hamster ◽  
Variable Number ◽  
Ovary Cell ◽  
Mrna Levels ◽  
Three Dimensional Model ◽  
Nucleotide Sugar ◽  
Sugar Transporters ◽  
Ovary Cell Line

AbstractSurvival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) fromTrypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line.TcNST2mRNA levels were detected in allT. cruzilife-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

scholarly journals Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells.

1985 ◽  
Vol 5 (12) ◽  
pp. 3525-3531 ◽  
Author(s):  
J K Griffith
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Recombinant Dna ◽  
Chinese Hamster ◽  
Single Copy ◽  
Ovary Cell ◽  
Gene Copy ◽  
Mrna Levels ◽  
Protein Levels ◽  
Copy Numbers

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.

Tolerance of glycosylphosphatidylinositol (GPI)-specific phospholipase D overexpression by Chinese hamster ovary cell mutants with aberrant GPI biosynthesis

2001 ◽  
Vol 361 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Xiaohan DU ◽  
Jiewei CAI ◽  
Jian-zhong ZHOU ◽  
Victoria L. STEVENS ◽  
Martin G. LOW
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase D ◽  
Chinese Hamster Ovary ◽  
Expression System ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Toxic Substances ◽  
Wild Type ◽  
Transfected Cells ◽  
Empty Vector

Mammalian glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is capable of releasing GPI-anchored proteins by cleavage of the GPI moiety. A previous study indicated that overexpression of GPI-PLD in mouse RAW 264.7 monocytes/macrophages could be cytotoxic, since survivors of stable transfections had enzymic activity no higher than untransfected cells [Du and Low (2001) Infect. Immun. 69, 3214–3223]. We investigated this phenomenon by transfecting bovine GPI-PLD cDNA stably into Chinese hamster ovary (CHO) cells using a bi-cistronic expression system. The surviving transfectants showed an unchanged cellular level of GPI-PLD, supporting the cytotoxicity hypothesis. However, when using a CHO mutant defective in the second step of GPI biosynthesis as host, the expression level of GPI-PLD in stable transfectants was increased by 2.5-fold compared with untransfected or empty-vector-transfected cells. To identify the mechanism, we studied another CHO cell mutant (G9PLAP.D5), which seems to be defective at a later stage in GPI biosynthesis. In sharp contrast with wild-type cells, GPI-PLD activity in G9PLAP.D5 transfected with bovine GPI-PLD cDNA was 100-fold higher than untransfected or empty-vector-transfected cells. This was accompanied by a significant release of alkaline phosphatase into the medium and a decrease in membrane-associated alkaline phosphatase. Taken together, our results indicate that overexpression of GPI-PLD is lethal to wild-type cells, possibly by catalysing the overproduction of GPI-derived toxic substances. We propose that cells with abnormal GPI biosynthesis/processing can escape the toxic effect of these substances.

scholarly journals Isolation of Chinese hamster ovary cell mutants requiring the continuous presence of taxol for cell division.

1983 ◽  
Vol 97 (1) ◽  
pp. 22-29 ◽  
Author(s):  
F R Cabral
Keyword(s):  
Cell Division ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Normal Growth ◽  
Time Lapse ◽  
Single Step ◽  
Ovary Cell ◽  
M Phase ◽  
Continuous Presence

Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D2O. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is approximately 10(-5) in a nonmutagenized population. Both alpha- and beta-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on two-dimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor.

Multiplex PCR Analysis of Ultraviolet A and B Induced Delayed and Early Mutations in V79 Chinese Hamster Cells

2004 ◽  
Author(s):  
Jostein Dahle ◽  
Paul Noordhuis ◽  
Trond Stokke ◽  
Debbie Hege Svendsrud ◽  
Egil Kvam
Keyword(s):  
Multiplex Pcr ◽  
Chinese Hamster ◽  
Pcr Analysis ◽  
Ultraviolet A ◽  
Chinese Hamster Cells
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