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Biomedical subjects

B Guo

Publications and source records attributed to B Guo.

At least 37 records · Page 2Linked to original sources
PubMed2000

Improving the performance of MALDI-TOF in oligonucleotide analysis using a new SDIFA technology.

A new technology termed SDIFA is developed to improve the mass resolution of linear matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in oligonucleotide analysis. Unlike the currently used delayed extraction method, SDIFA allows electrical isolation of the sample holder from ion extraction/acceleration and selectively samples part of the desorbed ions, thereby reducing the initial velocity distribution and improving resolution. In addition, a method was introduced to improve the space focusing of TOF. Isotope-limited mass resolution was obtained for oligonucleotides of up to 62 mer, and the true instrumental resolution reaches to 1,800 at 19.2 kDa. It was also demonstrated that excellent resolution was obtained across a large mass range using a single setting of acquisition parameters. This feature allows unambiguous identification of multiple A/T heterozygote samples in a mass range of 5,200-7,800 Da. Moreover, compared with DE, the performance of SDIFA was more stable, reproducible, and less dependent on the experimental conditions including the laser power, sample spots, sample substrates, delayed time, and extraction field strength. This enhanced ease of data acquisition is the key to automated spectrum acquisition.

Oligonucleotides↗
PubMed2000

CD2-mediated IL-12-dependent signals render human gamma delta-T cells resistant to mitogen-induced apoptosis, permitting the large-scale ex vivo expansion of functionally distinct lymphocytes: implications for the development of adoptive immunotherapy strategies.

The ability of human gamma delta-T cells to mediate a number of in vitro functions, including innate antitumor and antiviral activity, suggests these cells can be exploited in selected examples of adoptive immunotherapy. To date, however, studies to examine such issues on a clinical scale have not been possible, owing in large measure to the difficulty of obtaining sufficient numbers of viable human gamma delta-T cells given their relative infrequency in readily available tissues. Standard methods used to expand human T cells often use a combination of mitogens, such as anti-T-cell receptor antibody OKT3 and interleukin (IL)-2. These stimuli, though promoting the expansion of alpha beta-T cells, usually do not promote the efficient expansion of gamma delta-T cells. CD2-mediated, IL-12-dependent signals that result in the selective expansion of human gamma delta-T cells from cultures of mitogen-stimulated human peripheral blood mononuclear cells are identified. It is first established that human gamma delta-T cells are exquisitely sensitive to apoptosis induced by T-cell mitogens OKT3 and IL-2. Next it is shown that the CD2-mediated IL-12-dependent signals, which lead to the expansion of gamma delta-T cells, do so by selectively protecting subsets of human gamma delta-T cells from mitogen-induced apoptosis. Finally, it is demonstrated that apoptosis-resistant gamma delta-T cells are capable of mediating significant antitumor cytotoxicity against a panel of human-derived tumor cell lines in vitro. Both the biologic and the practical implications of induced resistance to apoptosis in gamma delta-T cells are considered and discussed because these findings may play a role in the development of new forms of adoptive cellular immunotherapy. (Blood. 2000;96:3827-3837)

Adult↗
PubMed2000

Bcl-G, a novel pro-apoptotic member of the Bcl-2 family.

A new member of the Bcl-2 family was identified, Bcl-G. The human BCL-G gene consists of 6 exons, resides on chromosome 12p12, and encodes two proteins through alternative mRNA splicing, Bcl-G(L) (long) and Bcl-G(S) (short) consisting of 327 and 252 amino acids in length, respectively. Bcl-G(L) and Bcl-G(S) have identical sequences for the first 226 amino acids but diverge thereafter. Among the Bcl-2 homology (BH) domains previously recognized in Bcl-2 family proteins, the BH3 domain is found in both Bcl-G(L) and Bcl-G(S), but only the longer Bcl-G(L) protein possesses a BH2 domain. Bcl-G(L) mRNA is expressed widely in adult human tissues, whereas Bcl-G(S) mRNA was found only in testis. Overexpression of Bcl-G(L) or Bcl-G(S) in cells induced apoptosis although Bcl-G(S) was far more potent than Bcl-G(L). Apoptosis induction by Bcl-G(S) depended on the BH3 domain and was suppressed by coexpression of anti-apoptotic Bcl-X(L) protein. Bcl-X(L) also coimmunoprecipitated with Bcl-G(S) but not with mutants of Bcl-G(S) in which the BH3 domain was deleted or mutated or with Bcl-G(L). Bcl-G(S) was predominantly localized to cytosolic organelles, whereas Bcl-G(L) was diffusely distributed throughout the cytosol. A mutant of Bcl-G(L) in which the BH2 domain was deleted displayed increased apoptotic activity and coimmunoprecipitated with Bcl-X(L), suggesting that the BH2 domain autorepresses Bcl-G(L).

Alternative Splicing↗
PubMed2000

A Saccharomyces gene family involved in invasive growth, cell-cell adhesion, and mating.

The cell wall of bakers' yeast contains a family of glycosyl-phosphatidylinositol (GPI)-linked glycoproteins of domain structure similar to the adhesins of pathogenic fungi. In wild-type cells each of these proteins has a unique function in different developmental processes (mating, invasive growth, cell-cell adhesion, or filamentation). What unifies these developmental events is adhesion, either to an inert substrate or to a cell. Although they differ in their specificities, many of these proteins can substitute for each other when overexpressed. For example, Flo11p is required during vegetative growth for haploid invasion and diploid filamentation, whereas Fig2p is required for mating. When overexpressed, Flo11p and Fig2p can function in mating, invasion, filamentation, and flocculation. The ability of Flo11p to supply Fig2p function in mating depends on its intracellular localization to the mating projection, where Fig2p normally functions in the adhesion of mating cells. Our data show that even distant family members retain the ability to carry out disparate functions if localized and expressed appropriately.

Cell Adhesion↗
PubMed2000

A new MALDI-TOF based mini-sequencing assay for genotyping of SNPS.

A new MALDI-TOF based mini-sequencing assay termed VSET was developed for genotyping of SNPs. In this assay, specific fragments of genomic DNA containing the SNP site(s) are first amplified, followed by mini-sequencing in the presence of three ddNTPs and the fourth nucleotide in the deoxy form. In this way, the primer is extended by only one base from one allele, while it is typically extended by two bases from another allele. The products are then analyzed using MALDI-TOF mass spectrometry. The genotype of the SNP site is identified based on the number of nucleotides added. This assay has been examined using both synthetic and genomic DNA samples. In addition, multiplexed assays were successfully performed to genotype four SNP sites in a single tube. The main aspect of this assay is that it can overcome the key problems associated with the currently used mini-sequencing methods. First, it significantly reduces the stringent high-resolution and extensive desalting requirements that are essential to the pinpoint assay. Second, it avoids the long extension problem associated with the PROBE assay.

DNA Mutational Analysis↗
PubMed2000

Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer.

CD105 (endoglin), a receptor for transforming growth factor (TGF) beta1 and beta3 in vascular endothelial cells, is highly up-regulated in blood vessels of tissues where neovascularisation occurs. It modulates endothelial-mesenchymal signalling and is essential for angiogenesis. Indeed, CD105 knock-out mice die from malvascularisation by 11.5 day p.c. In the present study CD105, TGFbeta1 and CD105/TGFbeta1 complexes were quantified in plasma samples from 77 healthy individuals and 92 patients with early stage breast cancer prior to any treatment. When compared with normal controls, both CD105 and CD105/TGFbeta1 complex levels were significantly elevated in breast cancer patients, whereas TGFbeta1 levels were lower in cancer patients. The most important finding to emerge was that CD105 levels were significantly increased in patients who developed distant metastasis compared with disease-free patients. While there was no significant difference between CD105 levels in controls compared to disease-free patients, it was significantly higher in patients with metastatic disease. Thus patients who had died following local relapse or distant metastases possessed the highest levels of CD105. Neither CD105/TGFbeta1 complex nor TGFbeta1 levels correlated with tumour progression. Our data indicate that CD105 might be a valuable novel angiogenic marker for identifying breast cancer patients who are at high risk of developing metastasis.

Adult↗
PubMed2000

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase.

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine.

Acetonitriles↗
PubMed2000

Matrix effects on selectivities of poly(ethylene glycol)s for alkali metal ion complexation in matrix-assisted laser desorption/ionization.

The effect of the matrix on poly(ethylene glycol) (PEG) cationization was investigated using an equimolar mixture of CsCl and LiCl as cationizing agents. It was observed that for both carboxylic acid and non-carboxylic acid matrices used, PEG cationization by a given alkali metal ion depends on the matrix used. For example, cesiated PEG ion intensities were much stronger than those of the corresponding lithiated PEG ions when equimolar concentrations of Cs(+) and Li(+) were present and IAA (indolacrylic acid) was the matrix. However, an opposite result was obtained when the same experimental conditions were utilized, but with HABA (2-(4-hydroxyphenylazo)benzoic acid) in place of IAA as the matrix.

Acrylates↗
PubMed2000

Structural effects on polyether cationization by alkali metal ions in matrix-assisted laser desorption/ionization

Structural effects on polyether cationization in matrix-assisted laser desorption/ionization (MALDI) are investigated using three different polyethers: PEG (polyethylene glycol), PPG (polypropylene glycol), and PTHF (polytetrahydrofuran). This study was performed using equimolar cesium and lithium chlorides as the cationizing agent. It was observed that the polyether structure variation led to a substantial change in polyether selectivity for alkali metal ion complexation. Moreover, it was found that like PEG, PPG displays a different selectivity for Cs+ and Li+ with different matrices. Discussion of these results and their implication in MALDI are given.

Journal Article↗
PubMed2000

Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex.

Pre-B cell receptor (pre-BCR) expression is critical for B lineage development. The signaling events initiated by the pre-BCR, however, remain poorly defined. We demonstrate that lipid rafts are the major functional compartment for human pre-B cell activation. A fraction of pre-BCR was constitutively raft associated, and receptor engagement enhanced this association. These events promoted Lyn activation and Igbeta phosphorylation and led to the generation of a raft-associated signaling module composed of tyrosine phosphorylated Lyn, Syk, BLNK, PI3K, Btk, VAV, and PLCgamma2. Formation of this module was essential for pre-BCR calcium signaling. Together, these observations directly link the previously identified genetic requirement for the components of this module in B lineage development with theirfunctional role(s) in human preBCR signaling.

B-Lymphocytes↗
PubMed2000

Characterization of protein kinase chk1 essential for the cell cycle checkpoint after exposure of human head and neck carcinoma A253 cells to a novel topoisomerase I inhibitor BNP1350.

Cellular topoisomerase I is an important target in cancer chemotherapy. A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity against human head and neck carcinoma A253 cells in vitro. As a basis for future clinical trials of BNP1350 in human head and neck carcinoma, in vitro studies were carried out to investigate its effect on DNA damage and cell cycle checkpoint response. The treatment of A253 cells with BNP1350 caused biphasic profiles of DNA fragmentation displayed from 0 to 48 h after 2-h exposure. Pulsed-field gel electrophoresis demonstrated that the first wave of DNA damage was mainly megabase DNA fragmentation, but the second wave of DNA damage was 50- to 300-kb DNA fragmentation in addition to megabase DNA damage. The cell cycle checkpoint response was characterized after exposure to 0.07 and 0.7 microM concentrations of BNP1350, the IC(50) and IC(90) values, respectively. After exposure to a low concentration of BNP1350 (IC(50)), A253 cells accumulated primarily in G(2) phase. In contrast, treatment with a high concentration of BNP1350 (IC(90)) resulted in S phase accumulation. The concentration-associated cell cycle perturbation by BNP1350 was correlated with different profiles of cell cycle-regulatory protein expression. When treated with the low concentration of BNP1350, cyclin B/cdc2 protein expression was up-regulated, whereas with the high concentration, no significant change was observed at 24 and 48 h. In addition, increased phosphorylation of a G(2) checkpoint kinase chk1 was observed when cells were treated with a low concentration of BNP1350, whereas only slight inhibition of chk1 activity was found in the cells treated with the higher concentration. Altered chk1 phosphorylation after DNA damage appears to be associated with specific phases of cell cycle arrest induced by BNP1350. Because A253 cells do not express the p53 protein, the drug-induced alterations of the G(2) checkpoint kinase chk1 are not p53-dependent.

CDC2 Protein Kinase↗
PubMed2000

Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.

The Runt related transcription factors RUNX (AML/CBF(alpha)/PEBP2(alpha)) are key regulators of hematopoiesis and osteogenesis. Using co-transfection experiments with four natural promoters, including those of the osteocalcin (OC), multi drug resistance (MDR), Rous Sarcoma Virus long terminal repeat (LTR), and bone sialoprotein (BSP) genes, we show that each of these promoters responds differently to the forced expression of RUNX proteins. However, the three RUNX subtypes (i.e. AML1, AML2, and AML3) regulate each promoter in a similar manner. Although the OC promoter is activated in a C terminus dependent manner, the MDR, LTR and BSP promoters are repressed by three distinct mechanisms, either independent of or involving the AML C terminus, or requiring only the conserved C-terminal pentapeptide VWRPY. Using yeast two hybrid assays we find that the C terminus of AML1 interacts with a Groucho/TLE/R-esp repressor protein. Co-expression assays reveal that TLE proteins repress AML dependent activation of OC gene transcription. Western and northern blot analyses suggest that TLE expression is regulated reciprocally with the levels of OC gene expression during osteoblast differentiation. Digital immunofluorescence microscopy results show that TLE1 and TLE2 are both associated with the nuclear matrix, and that a significant subset of each colocalizes with AML transcription factors. This co-localization of TLE and AML proteins is lost upon removing the C terminus of AML family members. Our findings indicate that suppression of AML-dependent gene activation by TLE proteins involves functional interactions with the C terminus of AML at the nuclear matrix in situ. Our data are consistent with the concept that the C termini of AML proteins support activation or repression of cell-type specific genes depending on the regulatory organization of the target promoter and subnuclear localization.

Animals↗
PubMed2000

Overexpression of Bax enhances antitumor activity of chemotherapeutic agents in human head and neck squamous cell carcinoma.

Overexpression of the Bax protein in human head and neck squamous cell carcinoma A253 cells was reported to result in an increased sensitivity to various chemotherapeutic agents in vitro (Guo et al., Oncol. Res., 11: 91-99, 1999). In the present study, the relationship between Bax expression and response to chemotherapy was further investigated in vitro and in vivo model systems. For in vitro study, A253, A253/Vec (pcDNA3 vector transfectant), and A253/Bax (pcDNA3/Bax transfectant, expressing 50-fold higher Bax protein than A253 and A253/Vec) cells were exposed to various concentrations of raltitrexed (a specific thymidylate synthase inhibitor) and SN-38 (a topoisomerase I inhibitor) for 2 h, and cell growth inhibition was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide clonogenic assay. Compared to A253/Vec, A253/Bax cells exhibited 9.5- and 13.5-fold increases in sensitivity to raltitrexed and SN-38, respectively. For in vivo study, A253/Vec and A253/Bax tumor xenografts were established by s.c. injection of tumor cells into nude mice. The antitumor activity and toxicity of raltitrexed (i.v. push daily for 5 days) and irinotecan (a prodrug of SN-38; i.v. push daily for 3 days) were evaluated. The maximum tolerated doses of raltitrexed and irinotecan were 30 and 100 mg/kg/day, respectively. At the maximum tolerated doses, minimal antitumor activity was observed with raltitrexed, although irinotecan was more active than raltitrexed against A253 or A253/Vec tumors. In contrast, both raltitrexed and irinotecan were significantly more active against A253/Bax xenografts than against A253/Vec xenografts; the yield for complete tumor regression (cure) was 40% and 100% with raltitrexed and irinotecan, respectively, with no significant toxicity. Furthermore, the observed increase of antitumor activity in A253/Bax tumors was associated with an enhanced induction of apoptosis in vivo. The in vivo results demonstrated a proof of the principal concept that selecting up-regulation of the proapoptosis gene Bax can provide the basis for a greater therapeutic efficacy to a variety of chemotherapeutic agents with different structures and mechanisms of action.

Animals↗
PubMed2000

[A comparative study on the capacities of different strains of Streptococcus sanguis for P-aminobenzoic acid production].

This study was intended to compare the capacities of different strains of Strep, sanguis for P-Aminobenzoic acid (PABA) production. The synthesis of PABA during the growth of four strains of Strep. sanguis was measured by the reversed-phase high-performance liquid chromatographic method. The results showed that the concentrations of PABA synthesized by S. sanguis 10556, S. sanguis 10557, S. sanguis S34 and S. sanguis H7-4. Were 1.979 +/- 0.081 micrograms/ml, 1.383 +/- 0.193 micrograms/ml, 1.983 +/- 0.052 micrograms/ml and 1.032 +/- 0.229 micrograms/ml, respectively, and in term of PABA concentration, S. sanguis 10556 was significantly different from S. sanguis 10557 and S. sanguis H7-4; S. sanguis S34 was significantly different from S. sanguis 10557 and S. sanguis H7-4. No significant difference was found between S. sanguis 10556 and S. sanguis S34, nor between S. sanguis 10557 and S. sanguis H7-4, either. In conclusion, the method is simple, rapid and accurate. S. sanguis did synthesize PABA, and the difference in ability for PABA formation existed among the four strains of S. sanguis. This study is helpful to researches on the symbiosis between S. sanguis and S. muntans and to determination of their role in the microbial homeostasis of dental plaque.

4-Aminobenzoic Acid↗
PubMed1999

Crystal structure of the nuclear matrix targeting signal of the transcription factor acute myelogenous leukemia-1/polyoma enhancer-binding protein 2alphaB/core binding factor alpha2.

Transcription factors of the acute myelogenous leukemia (AML)/polyoma enhancer-binding protein (PEBP2alpha)/core-binding factor alpha (CBFA) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. AML-1/PEBP2alphaB/CBFA2 proteins participating in transcription are associated with the nuclear matrix. This association is solely dependent on a highly conserved C-terminal protein segment, designated the nuclear matrix targeting signal (NMTS). The NMTS of AML-1 is physically distinct from the nuclear localization signal, operates autonomously, and supports transactivation. Our data indicate that the related AML-3 and AML-2 proteins are also targeted to the nuclear matrix in situ by analogous C-terminal domains. Here we report the first crystal structure of an NMTS in an AML-1 segment fused to glutathione S-transferase. The model of the NMTS consists of two loops connected by a flexible U-shaped peptide chain.

Amino Acid Sequence↗
PubMed1999

Involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-associated spontaneous apoptosis and differentiation in an A253 human head and neck carcinoma xenograft model.

Time-dependent ladder-type DNA fragmentation and morphological alterations consistent with apoptosis were observed among A253 human head and neck squamous cell carcinoma (HNSCC) cells in nude mice from 15 to 18 days after transplantation, without any drug treatment. No evidence of ladder-type DNA fragmentation was detected in A253 cells in vitro or in normal nude mouse tissues (skin and muscle). Our aim was to explore molecular factors associated with such spontaneous apoptosis. Bcl-2 protein expression decreased, while bax protein expression increased from day 9 after transplantation. Moreover, altered expression of bcl-2 and bax was accompanied by the increased proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Time-dependent dephosphorylation of Rb, followed by proteolytic cleavage, was also observed from day 9 after transplantation. The data indicate that the caspase-3 activation and cleavage of Rb protein may represent important steps in the regulation pathway of bax-mediated spontaneous apoptosis. Interestingly, the time-dependent activation of spontaneous apoptosis was almost simultaneous with the induction of differentiation and increased expression of several differentiation-associated regulatory proteins. An increased expression of cyclin D1 and cyclin-dependent kinase-5 (cdk5) was observed from day 9 after transplantation, whereas only slight alteration of cdk4 expression was found. The time-dependent activation of cyclin D1 and cdk5 preceded both the induction of ladder-type DNA fragmentation and increased keratin pearl formation. Furthermore, MCM3 was cleaved early in spontaneous apoptosis and differentiation. Our observations suggest the involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-mediated spontaneous apoptosis and differentiation in A253 xenografts. P53 and WAF1 proteins were not expressed in the xenografts, indicating that the changes in the regulatory proteins during apoptosis and differentiation were not p53 or WAF1 dependent.

Animals↗