Characterization of a Glycosyl Transferase Inactivating Macrolides, Encoded by gimA from Streptomyces ambofaciens

ABSTRACT In Streptomyces ambofaciens, the producer of the macrolide antibiotic spiramycin, an open reading frame (ORF) was found downstream of srmA, a gene conferring resistance to spiramycin. The deduced product of this ORF had high degrees of similarity to Streptomyces lividans glycosyl transferase, which inactivates macrolides, and this ORF was called gimA. The cloned gimA gene was expressed in a susceptible host mutant of S. lividans devoid of any background macrolide-inactivating glycosyl transferase activity. In the presence of UDP-glucose, cell extracts from this strain could inactivate various macrolides by glycosylation. Spiramycin was not inactivated but forocidin, a spiramycin precursor, was modified. In vivo studies showed that gimA could confer low levels of resistance to some macrolides. The spectrum of this resistance differs from the one conferred by a rRNA monomethylase, such as SrmA. InS. ambofaciens, gimA was inactivated by gene replacement, without any deleterious effect on the survival of the strain, even under spiramycin-producing conditions. But the overexpression of gimA led to a marked decrease in spiramycin production. Studies with extracts from wild-type and gimA-null mutant strains revealed the existence of another macrolide-inactivating glycosyl transferase activity with a different substrate specificity. This activity might compensate for the effect of gimA inactivation.

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NEGATIVE EFFECTS OF VITAMIN K PREPARATIONS ON GLUCURONYL TRANSFERASE ACTIVITY

PEDIATRICS ◽

10.1542/peds.40.6.993 ◽

1967 ◽

Vol 40 (6) ◽

pp. 993-999

Author(s):

Barbara Jones

Keyword(s):

Vitamin K ◽

Inhibitory Effect ◽

Water Soluble ◽

In Vivo Studies ◽

Transferase Activity ◽

Negative Effects ◽

Glucuronyl Transferase ◽

Glucuronide Conjugation

In vitro studies using a mouse liver microsome system failed to demonstrate that menadiol sodium diphosphate, menadione sodium bisulfite, or phytonadione enhanced or inhibited the quantity of ortho-aminophenol glucuronide produced. In vivo studies in young rats with these vitamin K analogues also failed to show an effect on glucuronide conjugation. Based on this data, it is concluded that the hyperbilirubinemia seen in prematures after large doses of water-soluble vitamin K analogues is probably not due to an inhibitory effect of glucuronyl transferase. The evidence suggesting that it may be due in part to hemolysis is briefly reviewed.

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Cell Surface Sialoglycopeptide Metabolism and Surface Glycosyl Transferase Activity in the Ehrlich Ascites Cell

Canadian Journal of Biochemistry ◽

10.1139/o75-122 ◽

1975 ◽

Vol 53 (8) ◽

pp. 895-902 ◽

Cited By ~ 5

Author(s):

D. Irwin ◽

T. P. Anastassiades

Keyword(s):

Sialic Acid ◽

Cell Surface ◽

Transferase Activity ◽

Intact Cells ◽

Neuraminidase Treatment ◽

Glycosyl Transferase ◽

Ehrlich Ascites ◽

Ehrlich Ascites Cell ◽

Almost All

1. The in vivo incorporation of radioactivity from [3H]glucosamine into a trypsin labile, cell surface sialoglycopeptide fraction (SGP) of Ehrlich ascites cells was studied in the presence and absence of puromycin pretreatment. The results indicated a much more complete inhibition of incorporation into the surface SGP than in the average intracellular acid insoluble glycoproteins. No evidence of turnover of the carbohydrate portion of the surface SGP independent of protein synthesis could be obtained.2. However, when intact cells were incubated with labelled uridine 5′-diphosphate – N-acetyl glucosamine or cytidine 5′-monophosphate (CMP) – sialic acid there was some incorporation largely into acid insoluble material, suggesting the presence of glycosyl transferase activity at the surface. Further evidence for surface activity was obtained when neuraminidase pretreatment of intact cells stimulated incorporation of labelled CMP – sialic acid sixfold and almost all of the incorporated counts could be released by subsequent neuraminidase treatment. Furthermore, a much greater proportion of the incorporated counts could be released by papain than by trypsin treatment of the intact cells. These results suggest that the surface acceptor for exogenously added CMP – sialic acid is not identical to the endogenously synthesized trypsin labile surface SGP.

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Structural Characterization of Three Novel Rat OKL38 Transcripts, Their Tissue Distributions, and Their Regulation by Human Chorionic Gonadotropin

Endocrinology ◽

10.1210/en.2004-0446 ◽

2004 ◽

Vol 145 (10) ◽

pp. 4763-4774 ◽

Cited By ~ 7

Author(s):

Choon Kiat Ong ◽

Chuan Young Ng ◽

Caine Leong ◽

Chee Pang Ng ◽

Chye Sun Ong ◽

...

Keyword(s):

Mammary Gland ◽

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Cellular Growth ◽

In Vivo Studies ◽

Protein Fusion ◽

Reading Frame ◽

Rat Mammary Gland

Abstract We previously identified a novel pregnancy-induced growth inhibitory gene, OKL38. To develop a rat model for further characterization of OKL38’s role in the initiation and progression of breast and ovarian cancer, we now report the cloning and characterization of three novel rat OKL38 cDNAs that are derived through alternative splicing and differential promoter usage. These three transcripts differ in their 5′ untranslated regions but share a common open reading frame that encoded for a 52-kDa protein. OKL38 is mapped to chromosome 19, spanning a region of approximately 15 kb, and contains eight exons. Differential expression of these three rat OKL38 transcripts was observed in liver, kidney, ovary, mammary gland, and uterus. In situ hybridization localized the rat OKL38 transcripts to the luminal epithelial cells of the rat mammary gland and to the granulosa cells in the rat ovary. In vivo studies showed that the RtOKL38-2.0 transcript and protein were regulated by human chorionic gonadotropin in the rat mammary gland and ovary. Importantly, overexpression of RtOKL38-enhanced green florescence protein fusion protein in Buffalo rat liver cells resulted in growth inhibition and cell death. Our present findings suggest that OKL38 may function as an effector for human chorionic gonadotropin protection against mammary carcinogenesis, and the availability of the three rat OKL38 cDNAs may help to elucidate the possible role of OKL38 in cellular growth, differentiation, and carcinogenesis.

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Secondary Structure Analysis of a Minimal Avian Leukosis-Sarcoma Virus Packaging Signal

Journal of Virology ◽

10.1128/jvi.74.1.456-464.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 456-464 ◽

Cited By ~ 35

Author(s):

Jennifer D. Banks ◽

Maxine L. Linial

Keyword(s):

Secondary Structure ◽

In Vivo Studies ◽

Packaging Signal ◽

Stem Loop ◽

Reading Frame ◽

Rous Sarcoma ◽

Secondary Structure Analysis ◽

Short Open Reading Frame ◽

Sarcoma Virus

ABSTRACT We previously identified a 160-nucleotide packaging signal, MΨ, from the 5′ end of the Rous sarcoma virus genome. In this study, we determine the secondary structure of MΨ by using phylogenetic analysis with computer modeling and heterologous packaging assays of point mutants. The results of the in vivo studies are in good agreement with the computer model. Additionally, the packaging studies indicate several structures which are important for efficient packaging, including a single-stranded bulge containing the initiation codon for the short open reading frame, uORF3, as well as adjacent stem structures. Finally, we show that the L3 stem-loop at the 3′ end of MΨ is dispensable for packaging, thus identifying an 82-nucleotide minimal packaging signal, μΨ, composed of the O3 stem-loop.

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Proteolysis of MLL Family Proteins Is Essential for Taspase1−Orchestrated Cell Cycle Progression.

Blood ◽

10.1182/blood.v108.11.769.769 ◽

2006 ◽

Vol 108 (11) ◽

pp. 769-769 ◽

Cited By ~ 2

Author(s):

James J. Hsieh ◽

Shugaku Takeda ◽

David Y. Chen ◽

Todd D. Westergard ◽

Jill K. Fisher ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Histone H3 ◽

Cancer Therapeutics ◽

In Vivo Studies ◽

Transferase Activity ◽

Methyl Transferase ◽

Cyclin E1

Abstract Taspase1 was identified as the threonine endopeptidase that cleaves MLL for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1−/− mice. Taspase1 deficiency results in non−cleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in cell cycle. Taspase1−/− animals are smaller in size. Taspase1−/− MEFs exhibit impaired proliferation and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down−regulation of Cyclin Es, As, and Bs and up−regulation of p16Ink4a. We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The un−cleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, CHIP assays demonstrate a markedly decreased histone H3 K4 tri−methylation at Cyclin E1 and E2 genes in Taspase1−/− cells. Furthermore, MLLnc/nc;2nc/nc MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1−/− cells are resistant to oncogenic transformation and Taspase1 is over−expressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.

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In vivo studies of catechol-o-methyl transferase activity following intoxication with ethanol, L-dopa and dopamine-derived alkaloids

Pharmacological Research Communications ◽

10.1016/s0031-6989(77)80024-6 ◽

1977 ◽

Vol 9 (2) ◽

pp. 203-214 ◽

Cited By ~ 4

Author(s):

A. Giovine ◽

M. Renis ◽

A. Bertolino

Keyword(s):

In Vivo Studies ◽

Transferase Activity ◽

Methyl Transferase

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PPARgamma inhibits GH synthesis and secretion and increases apoptosis of pituitary GH-secreting adenomas

Acta Endocrinologica ◽

10.1530/eje.0.1500863 ◽

2004 ◽

pp. 863-875 ◽

Cited By ~ 26

Author(s):

F Bogazzi ◽

F Ultimieri ◽

F Raggi ◽

D Russo ◽

R Vanacore ◽

...

Keyword(s):

Culture Media ◽

Ppar Gamma ◽

In Vivo Studies ◽

Gh3 Cells ◽

Peroxisome Proliferator ◽

Mrna Levels ◽

Dependent Manner ◽

Rt Pcr ◽

Cell Extracts

OBJECTIVE: The objective of the study was to evaluate the expression and functional activity of Peroxisome proliferator-activated receptor (PPAR) gamma in pituitary adenomas from 14 consecutive acromegalic patients and to establish its role in apoptosis. SUBJECTS AND METHODS: Fourteen consecutive acromegalic patients were enrolled in the study. Wistar-Furth rats were used for in vivo studies. Expression of PPARgamma was evaluated by RT-PCR and Western blot. Apoptosis and cell cycle were assessed by FACS analysis. The effects of PPARgamma ligands on transcriptional regulation of GH gene were evaluated by RT-PCR and electromobility shift assay. RESULTS: PPARgamma was expressed in all human GH-secreting adenoma (GH-oma), in normal pituitary tissue samples (39+/-24% and 78+/-5% of immunostained nuclei respectively; P<0.0002; ANOVA), and in rat GH-secreting (GH3) cells. A PPRE-containing reporter plasmid transfected into GH3 cells was activated by ciglitazone or rosiglitazone (TZDs), indicating that PPARgamma was functionally active. Treatment of GH3 cells with TZDs increased apoptosis in a dose-dependent manner (P=0.0003) and arrested cell proliferation, reducing the number of cells in the S-phase (P<0.0001 vs untreated cells). TZDs increased the expression of TRAIL, leaving unaffected that of p53 and Bax. TZDs reduced GH concentrations in the culture media from 43.7+/-5.4 ng/ml to 2.1+/-0.3 ng/ml (P<0.0001) and in cell extracts (P<0.004). PPARgamma-RXRalpha heterodimers bound to GH promoter, inhibiting its activity and reducing GH mRNA levels (1.8 x 10(6) vs 5.7 x 10(6) transcripts respectively vs untreated cells; P<0.002). Subcutaneous GH-oma developed in rats injected with GH3 cells; tumor growth increased in placebo-treated rats and to a lesser extent in TZDs-treated animals (24.1+/-2.0 g, and 14.8+/-4.2 g respectively, P<0.03). Serum GH concentrations were lower in TZDs-treated rats than in controls (871+/-67 ng/ml vs 1.309+/-238 ng/ml; P<0.05). CONCLUSIONS: The results of this study indicate that PPARgamma controls GH transcription and secretion as well as apoptosis and growth of GH-oma; thus, TZDs have the potential of a useful tool in the complex therapeutic management of acromegalic patients.

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