Biomedical subjects

Lin Li

Publications and source records attributed to Lin Li.

247 records · Page 14Linked to original sources

PubMed1999

Effects of Deleting the Amino-terminal Domain of GRK-2 on Its Function.

To reveal the possible role of the amino-terminal domain of G protein-coupled receptor kinases(GRKs)in receptor phosphorylation and/or modulation of its kinase activity, a truncated mutant of GRK-2 lacking the amino-terminal domain(deltaN-GRK2)was made. deltaN-GRK2 was expressed effectively in E.coli as a GST fusion protein and was purified by affinity chromatography on a GSH-Sepharose column. deltaN-GRK2 was then separated from GST tag by thrombin cleavage and recovered. Although deltaN-GRK2 had nearly identical activity with wild-type GRK-2 in phosphorylation of peptide substrate, it completely lost the ability to phosphorylate the light-activated receptor rhodopsin. Furthermore, deletion of the amino-terminal domain rendered GRK-2 unresponsive to the regulation of kinase activity by a truncated form of rhodopsin, (329)G-Rho(*) and beta gamma subunits of G protein. These results demonstrated that the amino-terminal domain was necessary to GRK2 for both the phosphorylation of receptor and the regulation of its kinase activity by the receptor. It was reasonable to postulate that this domain has little, if any effect on the catalytic domain of natural form of GRK2.

Journal Article↗

PubMed1999

Yeast Two-hybrid System and Its Application on Proteomics.

Proteomics is a new research area of the post-genomic era that aims at the analysis and identification of the entire proteins present in the cell, tissue or organism, and of the functions and the linkages of these proteins. Protein-protein interactions are characteristic of cellular activities and of course an important part of proteomics. One of the effective techniques to detect protein-protein interaction is yeast two-hybrid system. This system was successfully employed to analyze the protein linkage map of the yeast mRNA splicing machinery, which boded well for its application on human proteome research.

Journal Article↗

PubMed1997

The Preparation of the Antigen Conformation-related Monoclonal Antibodies against Maltose-Binding Protein.

Making use of the high expression efficiency of maltose-binding protein (MBP) in E. coli, the soluble and the inclusion body forms of MBP were purified to homogeneity by affinity chromatography and by denaturation, refolding and gel filtration respectively. After immunized Balb/C mice with two forms of MBP respectively, 5 clones for each antigen form were found to secrete monoclonal antibodies. The binding experiments showed that two antibodies obtained by the immunization with the refolded form of MBP had high affinity to the denatured MBP.

Journal Article↗

PubMed1996

Purification and Kinetic Studies of Yeast Alcohol Dehydrogenase I.

An efficient procedure for the purification of yeast alcohol dehydrogenase I (ADHI) was developed. By using Blue-Sepharose 4B affinity chromatography, ADHI from yeast (S. cerevisiae) was purified 200-fold with an overall yield of 47% to homogeneity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis and starch gel electrophoresis. Results of studies on the product and dead-end inhibition kinetics were in agreement with and ordered Bi Bi mechanism as proposed by Wratten and Cleland. Dioxane was found to be a competitive inhibitor of this enzyme. The complexity of the reaction mechanism of this enzyme is discussed.

Journal Article↗

PubMedDate not supplied

Human interleukin-5 expression is synergistically regulated by histone acetyltransferase CBP/p300 and transcription factors C/EBP, NF-AT and AP-1.

Interleukin-5 (IL-5) plays a central role in the growth and differentiation of eosinophils and contributes to several disease states including asthma. There has been considerable interest in the identification of the transcriptional mechanisms controlling the synthesis of this cytokine. The regulation of IL-5 message is primarily at the level of transcription and is likely to be controlled, to a large extent, by regulatory elements in the promoter region that can influence the transcriptional activity of the gene. In this study, we performed a series of transient transfection experiments with IL-5 promoter-reporter gene construct and expression plasmid for E1A, an inhibitor of CBP/p300, or for the CBP/p300-binding defective E1A Delta 2-36. The results showed that E1A repressed IL-5 promoter activity, while E1A Delta 2-36 had no effect on it. This suggested that CBP/p300 was involved in regulation of IL-5 gene expression. Transcriptional coactivator CBP/p300 and transcription factors C/EBP, NF-AT, and c-Fos synergistically activated IL-5 promoter. Furthermore, we found that ectopic expression of p300 increased endogenous IL-5 mRNA expression. Thus, the histone acetyltransferase (HAT) activity of CBP/p300 was required to activate IL-5 expression. This report provided evidence, for the first time, that CBP/p300 was involved in IL-5 gene expression and the HAT activity was important in regulation of IL-5 expression.

Acetyltransferases↗

PubMedDate not supplied

Vinyl sulfone bifunctional derivatives of DOTA allow sulfhydryl- or amino-directed coupling to antibodies. Conjugates retain immunoreactivity and have similar biodistributions.

We have synthesized a bifunctional vinyl sulfone-cysteineamido derivative of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that can be conjugated to the sulfhydryls of mildly reduced recombinant antibody (chimeric anti-CEA antibody cT84.66) at pH 7 or to the amino groups of lysine residues at pH 9. The conjugation is sulfhydryl specific at pH 7 (case 1), and amino specific at pH 9 (case 2) as long as the antibody has no free sulhydryl groups. At a molar ratio of 50 BCA (bifunctional chelating agent) to mAb, the number of chelates conjugated is 0.8 for case 1, and 4.6 for case 2. The resulting conjugates can be radiolabeled with (111)In to high specific activity (5 mCi/mg) with high efficiency (>95%) at 43 degrees C in 60 min. The radiolabeled conjugates retained >95% immunoreactivity and are stable in serum containing 1mM DTPA over 3 d. When the radiolabeled conjugates were injected into nude mice bearing LS174T human colon tumor xenografts, over 40% ID/g accumulated in tumors during the period 24-72h. Tumor-to-blood ratios were 4.5, 3.5, and 2.5 for the sulfhydryl coupled conjugate at 24, 48, and 72 h, respectively, and 2.7, 2.5, and 2.3 for the amino-coupled conjugate at the same time points. For other organs the biodistributions were nearly identical whether the conjugates were attached via sulfhydryl or amino groups. These novel BCAs are easy to synthesize, offer versatile conjugation options, and give equivalent biodistributions that result in high tumor uptake and good tumor-to-blood ratios.

Amino Acids↗

PubMedDate not supplied

Reduction of kidney uptake in radiometal labeled peptide linkers conjugated to recombinant antibody fragments. Site-specific conjugation of DOTA-peptides to a Cys-diabody.

Arano and co-workers (Arano et al. (1999) Cancer Res. 59, 128-134) have synthesized peptides with an N-terminal radioiodinated hippuric acid and a C-terminal lysine linked to antibody fragments via the epsilon-amino group of lysine that show reduced kidney uptake compared to antibody fragments directly radioiodinated. This approach takes advantage of the lysine specific carboxypeptidase activity of the kidney brush border enzymes that cleave off the radiolabeled peptide linker from the antibody fragment prior to uptake by proximal tubule cells. On the basis of their approach, we have synthesized a tetrapeptide with an N-terminal DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and a C-terminal (N(epsilon)-maleoyl)lysine that was site-specifically conjugated to an anti-CEA diabody (Yazaki et al. (2001) Bioconjugate Chem. 12, 220-228) that was engineered to contain a C-terminal cysteine (Cys-diabody). Biodistributions of the In-111-radiolabeled conjugate in nude mice show significantly reduced kidney uptake (a maximum of 82%ID/g at 6 h) compared to In-111 radiolabeled DOTA-diabody (184%ID/g at 6 h) in which DOTA was conjugated to endogenous lysine residues using DOTA-active ester chemistry. To further reduce kidney uptake, a homologous compound with a C-terminal (N(epsilon)-amino-1,6-hexane-bis-vinyl sulfone)lysine was synthesized and site-specifically conjugated to the Cys-diabody. Biodistributions of this In-111-labeled conjugate reduced kidney uptake to 54%ID/g at 6 h. To explore the effect of the relative positions of the chelate vs the cys-diabody on kidney uptake, we also synthesized a tetrapeptide with an N-terminal bromoacetate for conjugation to Cys-diabody and a C-terminal (N(epsilon)-amidino-propyl-3-thio-vinylsulfonyl-DO3A)lysine. This peptide essentially reverses the positions of the chelate and Cys-diabody attachment points on the peptide, while retaining the linker length on the epsilon-amino group of the lysine. In this case, biodistributions of the In-111-radiolabeled conjugate in nude mice showed high kidney uptake (189%ID/g at 6 h), comparable to that obtained with the In-111-radiolabeled active ester conjugated DOTA-diabody (184%ID/g at 6 h). We conclude that the peptide linker strategy of Arano and co-workers to reduce kidney uptake can be successfully applied to chelate/radiometal complexes and requires that the chelate/radiometal be located at the N-terminus of the peptide and the antibody fragment attachment site on the epsilon-amino group of the lysine. Furthermore, we demonstrated a role for the attachment chemistry to the epsilon-amino group of the lysine on the magnitude of kidney uptake.

Animals↗

PubMedDate not supplied

Improved biodistribution and radioimmunoimaging with poly(ethylene glycol)-DOTA-conjugated anti-CEA diabody.

Diabodies are single chain antibody fragments (scFvs) that spontaneously form bivalent dimers of molecular size 50-55000. Radiolabeled diabodies are almost ideal tumor targeting agents due to their high avidity (bivalent) binding to tumor antigens and small size (50-55000) that leads to improved tumor-to-blood ratio compared to intact antibodies (150000). However, due to their high retention and metabolism in the kidney, radioiodine is the current radiolabel of choice for diabodies since radioiodine is rapidly excreted from the kidney once metabolized. We have previously shown that 111In-DOTA-diabody gives higher tumor uptake in nude mouse xenografts than 125I-diabody, but has extremely high kidney retention since its 111In-labeled metabolites are retained by and only slowly excreted from the kidney. When a diabody is conjugated to a bifunctional PEG-3400 derivative followed by reaction with cysteinyl-DOTA, the resulting product has an apparent molecular size of 75000 and a Stokes radius of 35 angstroms on size exclusion chromatography, compared to a Stokes radius of 25 angstroms for intact diabody. When radiolabeled, the conjugate gives high yields of 111In-labeled product, retains high immunoreactivity, and gives improved biodistributions (30-40%ID/g, 12-48 h) compared to 111In-DOTA-diabody (12-13%ID/g, 6-12 h). We show that the improved biodistribution is due to an increase in Stokes radius caused by the linear PEG-3400 since conjugation of diabody with multiple (PEG)12 linkers followed by reaction with cysteinyl-DOTA does not reduce kidney accumulation. We also show that 111In-cysteinyl-DOTA-PEG3400-diabody gives excellent tumor images in the nude mouse xenograft model and that 125I-PEG3400-diabody gives equivalent images to 125I-minibody (molecular size, 80000), but improved tumor-to-liver ratios, suggesting that this imaging agent can be used to image liver metastases.

Animals↗

PubMedDate not supplied

Prevalence and evolution of drug resistance HIV-1 variants in Henan, China.

To understand the prevalence and evolution of drug resistant HIV strains in Henan China after the implementation of free antiretroviral therapy for AIDS patients. 45 drug naïve AIDS patients, 118 AIDS patients who received three months antiretroviral therapy and 124 AIDS patients who received six months antiretroviral treatment were recruited in the southern part of Henan province. Information on general condition, antiretroviral medicines, adherence and clinical syndromes were collected by face to face interview. Meanwhile, 14 ml EDTA anticoagulant blood was drawn. CD4/CD8 T cell count, viral load and genotypic drug resistance were tested. The rates of clinical improvement were 55.1% and 50.8% respectively three months and six months after antiretroviral therapy. The mean CD4 cell count after antiretroviral therapy was significantly higher than in drug naïve patients. The prevalence rate of drug resistant HIV strains were 13.9%, 45.4% and 62.7% in drug naïve patients, three month treatment patients and six month treatment patients, respectively. The number of resistance mutation codons and the frequency of mutations increased significantly with continued antiretroviral therapy. The mutation sites were primarily at the 103, 106 and 215 codons in the three-month treatment group and they increased to 15 codon mutations in the six-month treatment group. From this result, the evolution of drug resistant strains was inferred to begin with the high level NNRTI resistant strain, and then develop low level resistant strains to NRTIs. The HIV strains with high level resistance to NVP and low level resistance to AZT and DDI were highly prevalent because of the AZT+DDI+NVP combination therapy. These HIV strains were also cross resistant to DLV, EFV, DDC and D4T. Poor adherence to therapy was believed to be the main reason for the emergence and prevalence of drug resistant HIV strains. The prevalence of drug resistant HIV strains was increased with the continuation of antiretroviral therapy in the southern part of Henan province. Measures, that could promote high level adherence, provide new drugs and change ART regimens in failing patients, should be implemented as soon as possible.

CD4-Positive T-Lymphocytes↗

PubMedDate not supplied

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions.

Active host-pathogen interactions take place during infection of human immunodeficiency virus type 1 (HIV-1). Outcomes of these interactions determine the efficiency of viral infection and subsequent disease progression. HIV-infected cells respond to viral invasion with various defensive strategies such as innate, cellular and humoral immune antiviral mechanisms. On the other hand, the virus has also developed various offensive tactics to suppress these host cellular responses. Among many of the viral offensive strategies, HIV-1 viral auxiliary proteins (Tat, Rev, Nef, Vif, Vpr and Vpu) play important roles in the host-pathogen interaction and thus have significant impacts on the outcome of HIV infection. One of the best examples is the interaction of Vif with a host cytidine deaminase APOBEC3G. Although specific roles of other auxiliary proteins are not as well described as Vif-APOBEC3G interaction, it is the goal of this brief review to summarize some of the preliminary findings with the hope to stimulate further discussion and investigation in this exhilarating area of research.

HIV Infections↗

PubMedDate not supplied

Genetic variations of the porcine PRKAG3 gene in Chinese indigenous pig breeds.

Four missense substitutions (T30N, G52S, V199I and R200Q) in the porcine PRKAG3 gene were considered as the likely candidate loci affecting meat quality. In this study, the R200Q substitution was investigated in a sample of 62 individuals from Hampshire, Chinese Min and Erhualian pigs, and the genetic variations of T30N, G52S and V199I substitutions were detected in 1505 individuals from 21 Chinese indigenous breeds, 5 Western commercial pig breeds, and the wild pig. Allele 200R was fixed in Chinese Min and Erhualian pigs. Haplotypes II-QQ and IV-QQ were not observed in the Hampshire population, supporting the hypothesis that allele 200Q is tightly linked with allele 199V. Significant differences in allele frequencies of the three substitutions (T30N, G52S and V199I) between Chinese indigenous pigs and Western commercial pigs were observed. Obvious high frequencies of the "favorable" alleles 30T and 52G in terms of meat quality were detected in Chinese indigenous pigs, which are well known for high meat quality. However, the frequency of the "favorable" allele 199I, which was reported to have a greater effect on meat quality in comparison with 30T and 52G, was very low in all of the Chinese indigenous pigs except for the Min pig. The reasons accounting for this discrepancy remain to be addressed. The presence of the three substitutions in purebred Chinese Tibetan pigs indicates that the three substitutions were ancestral mutations. A novel A/G substitution at position 51 in exon 1 was identified. The results suggest that further studies are required to investigate the associations of these substitutions in the PRKAG3 gene with meat quality of Chinese indigenous pigs, and to uncover other polymorphisms in the PRKAG3 gene with potential effects on meat quality in Chinese indigenous pigs.

Adenine↗

PubMedDate not supplied

Hepatitis B knowledge and practices among Chinese immigrants to the United States.

INTRODUCTION: Chinese immigrants to the United States experience high rates of liver cancer. Chronic carriage of hepatitis B virus (HBV) is the most common underlying cause of liver cancer among Chinese Americans. Our objective was to describe Chinese immigrants' hepatitis B knowledge, testing, and vaccination levels. METHODS: A community-based, in-person survey of Chinese men and women was conducted in Seattle during 2005. Our study sample included 395 individuals. RESULTS: Less than one-half (48%) of our study group indicated they had received a hepatitis B blood test, and about one-third (31%) indicated they had been vaccinated against hepatitis B. The proportions of respondents who knew HBV can be spread during childbirth, during sexual intercourse, and by sharing razors were 70%, 54%, and 55%, respectively. Less than one-quarter of the study group knew that HBV cannot be spread by eating food that was prepared by an infected person (23%) and by sharing eating utensils with an infected person (16%). DISCUSSION: Over 50% of our respondents did not recall being tested for HBV. Important knowledge deficits about routes of hepatitis B transmission were identified. Continued efforts should be made to develop and implement hepatitis B educational campaigns for Chinese immigrant communities.

Adult↗

PubMedDate not supplied

Tobacco knowledge and beliefs in Chinese American men.

BACKGROUND: Chinese American men have relatively high smoking rates. However, there are limited data about the tobacco-related knowledge, attitudes, and beliefs of this racial/ethnic group. METHODS: We conducted a community-based telephone survey in Seattle, Washington during 2004. Households were identified by applying a previously validated list of Chinese last names to an electronic version of the Seattle telephone book. Interviews were completed in Cantonese, Mandarin, or English. Survey items addressed tobacco knowledge, cultural beliefs, and practices. RESULTS: The study sample included 168 Chinese American men. Current, former, and never smoking rates were 22%, 42%, and 36%, respectively. Current smokers were less likely to be proficient in English than never smokers, and were less likely to have a regular doctor than former smokers. They also had lower levels of knowledge about the health effects of tobacco, and were more likely to have traditional Chinese cultural beliefs about tobacco use than non-smokers. CONCLUSION: Tobacco use continues to be a public health problem among Chinese American men. Smoking cessation programs should target men with limited English proficiency and those without a regular source of health care. Educational materials should specifically address the negative health effects of smoking. They should also both acknowledge and address Chinese cultural beliefs about tobacco use.

Asian↗