Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio

Tsao, Jonglin; Lin, Xiujun; Lackland, Henry; Tous, Guillermo; Wu, Yifu; Stein, Stanley J.

doi:10.1016/0003-2697(91)90369-5

Cited by 13 publications

(8 citation statements)

References 10 publications

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“…The degree to which the peptides were conjugated was determined by loss of thiol groups in the peptide using Ellman's reagent (Pierce Chemical Co.). The extent of conjugation (typically 50-80 g peptide per mg of KLH) compared favorably with that previously seen for the attachment of peptides to KLH (14). For study of the induction of eosinophilia, 250 g of each peptide-KLH conjugate was adjuvanted with 20 g QS-21 in 0.1 ml of PBS and injected intramuscularly at 0 and 4 wk.…”

Section: Methodsmentioning

confidence: 83%

Atypical Pulmonary Eosinophilia Is Mediated by a Specific Amino Acid Sequence of the Attachment (G) Protein of Respiratory Syncytial Virus

Tebbey¹,

Hagen²,

Hancock³

1998

The Journal of Experimental Medicine

126

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We analyzed the immune responses evoked by a series of overlapping peptides to better understand the molecular basis for respiratory syncytial virus (RSV) G protein–induced eosinophilia in BALB/c mice. In vitro stimulation of spleen cells from natural G protein–primed mice showed dominant proliferative and cytokine (interferon [IFN]-γ and interleukin [IL]-5) responses to a peptide encompassing amino acids 184–198. Mice vaccinated with peptide 184– 198 conjugated to keyhole limpet hemocyanin showed significant pulmonary eosinophilia (39.5%) after challenge with live RSV. In contrast, mice immunized with a peptide (208–222) conjugate associated with induction of IFN-γ secreting spleen cells did not exhibit pulmonary eosinophilia after challenge. The in vivo depletion of CD4+ cells abrogated pulmonary eosinophilia in mice vaccinated with the peptide 184–198 conjugate, whereas the depletion of CD8+ cells had a negligible effect. Therefore, we have identified an association between peptide 184– 198 of natural G protein and the CD4+ T cell–mediated induction of pulmonary eosinophilia after live RSV challenge. Out of 43 human donors, 6 provided peripheral blood mononuclear cells that showed reactivity to G protein from RSV A2, 3 of which responded to peptide 184– 198. The results have important implications for the development of a vaccine against RSV.

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Section: Methodsmentioning

confidence: 83%

Atypical Pulmonary Eosinophilia Is Mediated by a Specific Amino Acid Sequence of the Attachment (G) Protein of Respiratory Syncytial Virus

Tebbey¹,

Hagen²,

Hancock³

1998

The Journal of Experimental Medicine

126

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“…Polyclonal antisera to hyperacetylated H4 were raised by immunization with a synthetic peptide corresponding to residues 2-19 of histone H4 and acetylated in the ε-amino groups of lysines 5, 8, 12, and 16. The acetylated peptide was custom-synthesized by Genosys and coupled to keyhole limpet hemocyanin (20). The immunoglobulin (IgG) fraction was purified by HiTrap protein-A FPLC chromatography according to the manufacturer (Amersham Pharmacia Biotech).…”

Section: Production Of Antisera To Hyperacetylated Histone H4 and Antmentioning

confidence: 99%

Liver‐specific methionine adenosyltransferaseMAT1Agene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications forMAT1Asilencing during transformation

et al. 2000

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Methionine adenosyltransferase (MAT) is the enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main donor of methyl groups in the cell. In mammals MAT is the product of two genes, MAT1A and MAT2A. MAT1A is expressed only in the mature liver whereas fetal hepatocytes, extrahepatic tissues and liver cancer cells express MAT2A. The mechanisms behind the tissue and differentiation state specific MAT1A expression are not known. In the present work we examined MAT1A promoter methylation status by means of methylation sensitive restriction enzyme analysis. Our data indicate that MAT1A promoter is hypomethylated in liver and hypermethylated in kidney and fetal rat hepatocytes, indicating that this modification is tissue specific and developmentally regulated. Immunoprecipitation of mononucleosomes from liver and kidney tissues with antibodies mainly specific to acetylated histone H4 and subsequent Southern blot analysis with a MAT1A promoter probe demonstrated that MAT1A expression is linked to elevated levels of chromatin acetylation. Early changes in MAT1A methylation are already observed in the precancerous cirrhotic livers from rats, which show reduced MAT1A expression. Human hepatoma cell lines in which MAT1A is not expressed were also hypermethylated at this locus. Finally we demonstrate that MAT1A expression is reactivated in the human hepatoma cell line HepG2 treated with 5-aza-2'-deoxycytidine or the histone deacetylase inhibitor trichostatin, suggesting a role for DNA hypermethylation and histone deacetylation in MAT1A silencing.

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“…The acetylated peptide was custom-synthesized by Genosys and coupled to Keyhole Limpet Hemocyanin [24]. The IgG fraction was puri®ed from the serum by HiTrap Protein-A FPLC chromatography according to the manufacturer (Amersham Pharmacia Biotech).…”

Section: Production Of Antisera To Hyperacetylated Histone H4 and Antmentioning

confidence: 99%

DNA methylation and histone acetylation of rat methionine adenosyltransferase 1A and 2A genes is tissue-specific

Torres

López-Rodas

Latasa

et al. 2000

The International Journal of Biochemistry & Cell Biology

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Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet). In mammals MAT activity derives from two separate genes which display a tissue-speci®c pattern of expression. While MAT1A is expressed only in the adult liver, MAT2A is expressed in non-hepatic tissues. The mechanisms behind the selective expression of these two genes are not fully understood. In the present report we have evaluated MAT1A and MAT2A methylation in liver and in other tissues, such as kidney, by methylationsensitive restriction enzyme digestion of genomic DNA. Our data indicate that MAT1A is hypomethylated in liver and hypermethylated in non-expressing tissues. The opposite situation is found for MAT2A. Additionally, histones associated to MAT1A and MAT2A genes showed enhanced levels of acetylation in expressing tissues (two-fold for MAT1A and 3.5-fold for MAT2A liver and kidney respectively). These observations support a role for chromatin structure and its modi®cation in the tissue-speci®c expression of both MAT genes. #

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Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio

Cited by 13 publications

References 10 publications

Atypical Pulmonary Eosinophilia Is Mediated by a Specific Amino Acid Sequence of the Attachment (G) Protein of Respiratory Syncytial Virus

Atypical Pulmonary Eosinophilia Is Mediated by a Specific Amino Acid Sequence of the Attachment (G) Protein of Respiratory Syncytial Virus

Liver‐specific methionine adenosyltransferaseMAT1Agene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications forMAT1Asilencing during transformation

DNA methylation and histone acetylation of rat methionine adenosyltransferase 1A and 2A genes is tissue-specific

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