Biomedical subjects
Publications and source records attributed to Y Guo.
A series of succinyl hydroxamate MMP inhibitors were prepared incorporating an aryl amino ketone moiety in place of the more typical C-terminal amino acid amides. Compounds of the C-terminal ketone series displayed potent inhibition of MMPs. Several compounds of the series were shown to be orally bioavailable.
Interference with polyamine transport and biosynthesis has emerged as an important anticancer strategy involving polyamine analogues and specific inhibitors of key biosynthetic enzymes. Because the prostate gland has a high polyamine content, by using the polyamine transporter for selective uptake into cancer cells, alkylating polyamines are likely to be highly effective against prostatic tumors. We have recently synthesized a novel class of spermine analogues, the lead compound of which has efficacy against human cancer cells (P. S. Callery et al., U. S. patent, 5,612,239, Issued March 17, 1997.). In this study, to investigate the potential therapeutic efficacy of the lead spermine analogue 1,12-diaziridinyl-4, 9-diazadodecane (BIS), against advanced prostate cancer, we examined the in vitro effect and in vivo efficacy of the compound in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. BIS exhibited a dose-dependent cytotoxic effect against prostate cancer cells via induction of apoptosis. Treatment of cells with BIS (1 microM) for 24 h resulted in a significant induction of apoptosis (24%). Exposure of BIS-treated PC-3 prostate cancer cells to gamma-irradiation resulted in a significant increase in the number of cells undergoing apoptosis and a subsequent decrease in the IC50. Furthermore, BIS treatment led to a significant enhancement of loss of clonogenic survival in irradiated prostate cancer cells (both PC-3 and DU-145). In vivo efficacy trials demonstrated a significant antitumor effect of BIS against both PC-3 and DU-145 tumor xenografts in severe combined immunodeficient mice in a dose-dependent pattern at maximally tolerated doses. Terminal transferase end-labeling analysis indicated that BIS-mediated tumor regression in vivo occurs via induction of apoptosis among prostatic tumor cells. These results suggest that the novel spermine analogue BIS: (a) has a potent antitumor effect against prostatic tumors via induction of apoptosis; and (b) increases the radiosensitivity of human prostate cancer cells by decreasing the apoptotic threshold to radiation. This study may have important clinical implications for the manipulation of this antitumor activity of the polyamine analogue for the optimization of the therapeutic efficacy of radiation in patients with advanced prostate cancer.
The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand. Opening is tightly regulated, but how it occurs is not known. These experiments identify an activity, recognition of specific DNA fork junctions, and suggest that it is critical to bacterial promoter opening. This activity is both sequence and structure specific; it recognizes the bases that constitute the upstream double-stranded/single-stranded boundary of the open complex. Promoter mutations known to reduce opening rates lead to comparable reductions in fork junction binding affinity. The activity acts to establish the upstream boundary of melted DNA and works in conjunction with two single-stranded DNA binding activities that recognize separately the two melted strands. The junction binding activity is contained within the sigma factor component of the holoenzyme. The activity occurs in both a typical prokaryotic transcription system and in a eukaryotic-like bacterial system that responds to enhancers and needs ATP. Thus DNA opening catalyzed by fork junction binding may occur in a variety of systems in which DNA must be opened to be copied.
The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five-stranded coiled-coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2-10 degreesC. For all-trans retinol and all-trans retinoic acid, an equilibrium dissociation constant KD = 0.6 microM was evaluated by fluorescence titration. Binding of benzene and all-trans retinol into the hydrophobic axial pore of the COMP coiled-coil domain was proven by the X-ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled-coil domain. The retinol beta-ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all-trans retinol with affinity similar to the wild-type, demonstrating that hydrophobic interactions are predominant.
Heat shock and other proteotoxic stresses cause accumulation of nonnative proteins that trigger activation of heat shock protein (Hsp) genes. A chaperone/Hsp functioning as repressor of heat shock transcription factor (HSF) could make activation of hsp genes dependent on protein unfolding. In a novel in vitro system, in which human HSF1 can be activated by nonnative protein, heat, and geldanamycin, addition of Hsp90 inhibits activation. Reduction of the level of Hsp90 but not of Hsp/c70, Hop, Hip, p23, CyP40, or Hsp40 dramatically activates HSF1. In vivo, geldanamycin activates HSF1 under conditions in which it is an Hsp90-specific reagent. Hsp90-containing HSF1 complex is present in the unstressed cell and dissociates during stress. We conclude that Hsp90, by itself and/or associated with multichaperone complexes, is a major repressor of HSF1.
It is generally believed that CTLs mature in lymphoid organs and then migrate into target tissues to execute their effector functions. This notion, however, is based on studies using antigens that are readily localized in the lymphoid tissue, such as viruses and allogeneic transplants. The site for maturation of CTLs for nonmetastatic tumors has not been determined. Because nonmetastatic tumor cells are not localized in lymphoid tissues, it is questionable whether such tumors are efficient inducers of antitumor CTLs. Here, we report that a nonmetastatic B7+ plasmacytoma induces strong effector CTL response. Thus, it is possible to induce CTLs with strong ex vivo CTL activity in the absence of tumor metastasis. In addition, a detailed kinetic analysis of CD8 T cell recruitment and maturation of CTL activity suggests that antitumor CTLs mature within the tumor rather than in the lymphoid tissues. Interestingly, despite B7-1 expression on tumor cells, induction of effector CTLs also requires costimulation by B7 on host antigen-presenting cells. These findings have important implications for tumor gene therapy and for understanding the mechanism of CTL induction in vivo.
The goal of this study was to test the hypothesis that the cardioprotective effects of the late phase of ischemic preconditioning (PC) can be mimicked by treatment with NO donors. In phase I (studies of myocardial stunning), conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In group I (controls, n=6), the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 54% on days 2 and 3 compared with day 1 (P<0.05), indicating a late PC effect against myocardial stunning. When rabbits were given the NO donors diethylenetriamine/NO (DETA/NO, 0.1 mg/kg i.v., 4 times [group II, n=5]) or S-nitroso-N-acetylpenicillamine (SNAP, 2.5 microg x kg(-1) x min(-1) i.v. for 75 minutes [group III, n=51) 24 hours before the first sequence of occlusion/reperfusion cycles, the deficit of WTh on day 1 was 60% (group II) and 54% (group III) less than that observed in controls (P<0.05 for both). In both groups II and III, there was no further improvement in the deficit of WTh on days 2 and 3 compared with day 1. The protective effect of DETA/NO was completely abrogated when this agent was given in conjunction with the ONOO- and .OH scavenger mercaptopropionyl glycine (MPG) (group IV, n=5). In phase II (studies of myocardial infarction), conscious rabbits underwent a 30-minute coronary occlusion followed by 3 days of reperfusion. When rabbits were preconditioned 24 hours earlier with six 4-minute occlusion/4-minute reperfusion cycles, infarct size was reduced by 43% (33.2+/-2.7% versus 58.3+/-4.1% of the region at risk in controls, P<0.05), indicating a late PC effect against myocardial infarction. When rabbits were pretreated with DETA/NO (group VII, n=8) or SNAP (group IX, n=7) 24 hours before the 30-minute occlusion, infarct size was reduced by a similar degree (29.3+/-3.6% and 32.0+/-3.3% of the region at risk, respectively; P<0.05 versus controls). The degree of protection could not be increased by doubling the dose of DETA/NO (group VIII, n=5). Coadministration of MPG completely abrogated the infarct-sparing action of DETA/NO (group X, n=7). Taken together, these results demonstrate that in conscious rabbits the administration of 2 structurally unrelated NO donors induces protection 24 hours later against both reversible (stunning) and irreversible (infarction) ischemia/reperfusion injury and that the magnitude of this protection is indistinguishable from that observed during the late phase of ischemic PC. The fact that the late phase of ischemic PC can be mimicked by NO donors provides direct evidence that NO in itself is sufficient to elicit this cardioprotective mechanism. The fact that NO donor-induced late PC was abrogated by MPG indicates that the mechanism whereby NO induces this phenomenon involves the generation of oxidant species, possibly ONOO- and/or .OH. Since a relatively brief treatment with hemodynamically inactive doses of NO donors can induce long-lasting protective effects, these agents could be useful for preconditioning the heart in patients.
Peripherin/rds plays an essential role in the maintenance of photoreceptor rod cell disk membrane structure. The purification of this protein to homogeneity [Boesze-Battaglia, K., et al. (1997) Biochemistry 36, 6835-6846] has allowed us to characterize the functional role of peripherin/rds in the maintenance of rod outer segment (ROS) membrane fusion processes. Utilizing a cell-free fusion assay system, we report that the fusion of R18-labeled ROS plasma membrane (R18-PM) with disk membranes or peripherin/rds-enriched large unilammellar vesicles (LUVs) is inhibited upon trypsinolysis of peripherin/rds. To understand this phenomenon, we tested the ability of a series of overlapping synthetic C-terminal peripherin/rds peptides to mediate model membrane fusion. Within the 63 amino acid long region of the C-terminus, we identified a minimal 15 residue long amino acid sequence (PP-5), which is necessary to promote membrane fusion. PP-5 was able to inhibit R18-PM disk membrane fusion and promoted ANTS/DPX contents mixing in a pure vesicle system. This peptide (PP-5) promoted calcium-induced vesicle aggregation of phosphatidylethanolamine:phosphatidylserine LUVs. FTIR analysis confirmed the structural prediction of this peptide as alpha-helical. When modeled as an alpha-helix, this peptide is amphiphilic with a hydrophobicity index of 0.75 and a hydrophobic moment of 0.59. PP-5 has substantial biochemical and functional homology with other well-characterized membrane fusion proteins. These results demonstrate the necessity for peripherin/rds in ROS membrane fusion, specifically the requirement for an intact C-terminal region of this protein.
Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification.
OBJECTIVE: Enzyme assays for the diagnosis of lysosomal storage diseases have been underway in China only in a few laboratories, and the specimens for enzyme assay must not be inactivated by environmental factors. To solve the problem, the stability of freeze-drying lysosomal enzymes was tested. METHODS: Three pools for samples were set up: control leucocytes pool and plasma pool(40 individuals in each pool), control fibroblasts pool( 3 cell lines). 4 groups of different dispositions of samples were designed for the pools:(1)pellets or plasma directly stored at -80 degrees C as routine(non-freeze-drying group or N);(2)after freeze-drying, pellets or plasma immediately stored at -80 degrees C (0 week or 0W); (3) after freeze-drying, pellets or plasma stayed at room temperature for 1 week(simulated mail) then stored at -80 degrees C (1 week or 1W);(4) the same with 1W but 3 weeks at room temperature(3 weeks or 3W). Microassay for enzyme activity was employed: 20 enzymes in leucocytes pool, 14 enzymes in fibroblasts pool and 7 in plasma pool. RESULTS: Enzyme activities among 4 groups in each sample pool were compared. Single factor analysis of variance (F test) showed no significance of differences (P>0.05) among 4 groups in leucocytes and fibroblasts pool, but highly significant differences (P<0.01) among 4 groups in plasma pool. "t test" showed no significance of differences (P> 0.05) between N and 3W in leucocytes and also fibroblasts pool, but very significant differences (P<0.01) between N and 3W in plasma pool. CONCLUSION: The results suggest that freeze-drying samples from leucocytes and fibro- blasts can provide a stable resource for enzyme assay and be simple for transportation of the diagnostic samples. Though freeze-drying is not appropriate for the plasma samples, it is still applicable for wide use since only a few kinds of lysosomal enzyme being indicated to use plasma for enzyme assay.
Expression of B7-family costimulatory molecules CD80 (B7-1) and CD86 (B7-2) on tumor cells enhances host immunity. However, the role of the two B7 receptors, CD28 and CTLA4 (CD152), on T cells in antitumor immune response has not been clearly elucidated. Based on the effects of anti-CD28 and anti-CTLA4 mAbs on T cell response, it was proposed that CD28-B7 interaction promotes antitumor immunity, whereas B7-CTLA4 interaction down-regulates it. A critical test for the hypothesis is whether selective engagement of CTLA4 receptors by their natural ligands CD80 and CD86 enhances or reduces antitumor immunity. Here we used tumors expressing wild-type and mutant CD80, as well as mice with targeted mutation of CD28, to address this issue. We report that in syngeneic wild-type mice, B7W (W88>A), a CD80 mutant that has lost binding to CD28 but retained binding to CTLA4, can enhance the induction of antitumor cytotoxic T lymphocytes (CTL); B7Y (Y201>A), which binds neither CD28 nor CTLA4, fails to do so. Consistent with these observations, B7W-transfected J558 plasmocytoma and EL4 thymoma grow significantly more slowly than those transfected with either vector alone or with B7Y. Optimal tumor rejection requires wild-type CD80. Moreover, expression of a high level of CD80 on thymoma EL4 cells conveys immunity in mice with a targeted mutation of CD28 gene. Taken together, our results demonstrate that B7-CTLA4 interaction enhances production of antitumor CTL and resistance to tumor challenge and that optimal enhancement of antitumor immunity by CD80 requires its engagement of both CD28 and CTLA4.
The amino acid sequence of full-length ovine fibroblast growth factor-1 (FGF-1) was determined by a combination of protein and cDNA sequencing. FGF-1 cDNA analysis indicated that ovine kidney cells express mRNAs encoding both full-length FGF-1 and a truncated FGF-1 variant. An overall comparison of the ovine FGF-1 primary sequence to the eight species studied to date revealed a high degree of conservation, with ovine FGF-1 sharing 90 and 95% sequence identity with human FGF-1 and bovine FGF-1, respectively. Additionally, the FGF-1 proteins from the various species have conserved cysteine residues at positions 30 and 97 and contain acetylated amino-terminal alanine residues. Mass spectrometry analysis confirmed that the blocking group of ovine FGF-1 is also consistent with that of an acetyl-moiety. In contrast to the other FGF-1 proteins, the 154 residue primary sequence of ovine FGF-1 contains three unique amino acid differences: Arg9, Arg44, and Ile123. Ovine FGF-1, unlike human FGF-1, is a potent mitogenic factor for NIH 3T3 fibroblasts in the absence of heparin. In the presence of exogenous heparin, the mitogenic activity of ovine FGF-1 is potentiated slightly.
Mapping of tryptic phosphopeptides of heat shock factor 1 (HSF1) from non-stressed or moderately heat-stressed HeLa cells, labeled in vivo by [32P]orthophosphate, revealed four major phosphopeptides A to D. Heat stress drastically increased phosphopeptide signals. To identify target peptides and amino acids and to correlate phosphorylation and transactivation function, phosphopeptide maps were produced of LexA-human HSF1 chimeras and mutant derivatives thereof, and transactivation activities of original and mutant chimeras were compared. LexA-HSF1 chimeras were previously shown to be regulated identically to HSF1, except that they transactivate promoters with LexA-binding sites instead of hsp promoters. The patterns of phosphopeptides of LexA-HSF1 and endogenous HSF1 were similar. Analysis of single residue substitutions suggested that phosphopeptide C is peptide VKEEPPSPPQSPR (297-309) phosphorylated on Ser-307 but not Ser-303. Substitution of Ser-307 but not Ser-303 caused deregulation of factor activity. Mapping of several constitutively active chimeras associated unphosphorylated peptide C with the transcriptionally active HSF1 conformation, suggesting that dephosphorylation of this peptide (at Ser-307) may either be an integral step in the activation process or serve to maintain the active conformation of HSF1. Exploiting this correlation, indirect evidence was obtained that activation domains of HSF1 interact with the distantly located regulatory domain to maintain the factor in an inactive state.
OBJECTIVE: This was a retrospective analysis of cytogenetic data from 600 cases with chronic myelogenous leukemia (CML) to investigate the features of Ph chromosome and its significance. METHODS: Bone marrow direct method and/or short-term culture were used to prepare the chromosomes and karyotype analysis was performed with R-banding technique. RESULTS: 30 cases (5%) were Ph negative; 570 cases (95%) were Ph positive. 535 cases (93.8%) had standard Ph translocation;34 cases(5.9%) had variant translocation, including 13 cases (2.2%) with simple variant translocation, 13 cases (2.2%) with complex variant translocation and 8 cases (1.4%) with masked Ph chromosome. 526 cases (92.2%) had 100% of Ph positive cells; 44 cases (7.7%) had normal karyotype in partial or all metaphases after treatment such as allogeneic bone marrow transplantation, interferon and pulse hydroxyurea therapy, but conventional chemotherapy had no effect on the percentage of Ph positive cells. 50.6% of Ph positive CML with blast crisis had extra chromosomal abnormalities, of which, the most common ones were +8(46.1%),2 Ph(33.9%) and i(17q) (23%) in descending order. CONCLUSION: These facts indicate that chromosome examinations not only help diagnose and differentiate CML,but also help predict the blast crisis, evaluate the therapeutic effect, and make a cytogenetic classification for CML.
Five novel polyacetylenes (5-9) were isolated from two different populations of the sponge Petrosia ficiformis collected in the Mediterranean Sea and the Atlantic Ocean. Their structures were established by extensive NMR analysis and by comparison with known petroformynes.
OBJECTIVE: To explore genetic patterns of idiopathic epilepsy (IEP). METHODS: Using familial analysis, tests for multifactorial inheritance and segregation analysis, we studied 210 pedigrees with IEP found in a population survey in Shangdong province. RESULTS: The genetic pattern of IEP is not polygenic but is mainly influenced by autosomal recessive disorders. The results of segregation analysis indicate that the genetic pattern of U*U multiplex families and U*A group is autosomal recessive. Only a few cases in U*U group may accept the assumption of autosomal recessive inheritance while the other are sporadic cases. The frequency of sporadic cases is approximately 78.5%. Genetic heterogeneity may influence U*U(f) group and U*U group. CONCLUSION: Further and careful empirical scrutiny of U*U(f) group and the sporadic cases in U*U group offers the best hope for getting a clear understanding of genetic patterns and mechanisms in IEP.
In this article, we have presented an overview of emerging novel techniques for early-light transillumination imaging as well as nonlinear optical tomography of body organs. The use of light for probing and imaging biomedical media offers the promise for development of safe, noninvasive, and inexpensive clinical imaging modalities with diagnostic ability. The strong scattering of light by biological tissues buries the shadowgram formed by forward-propatating image-bearing photons in the background noise of multiple-scattered light. Several methods for extraction of image-bearing light that capitalize on spatial, temporal and polarization characteristics of transmitted light are reviewed. More recently emerging nonlinear-optical histopathology methods for imaging subsurface structures of tissues in terms of its local spatial symmetry and molecular content are introduced. The progress made so far indicates that some of these techniques are apt to make a transition from laboratory to useful clinical modalities.
A mixed-mode separation technique has been developed and optimized for the separation of the 16 Environmental Protection Agency (EPA) priority polycyclic aromatic hydrocarbons (PAHs). The procedure utilized two different buffer additives as pseudo-stationary phases with different selectivities towards the analytes. Sodium dioctyl sulfosuccinate (DOSS) displayed selectivities for PAHs which were somewhat similar to the C18 phase in reversed-phase high performance liquid chromatography (HPLC). High acetonitrile content required for an effective separation prevented the formation of micelles as confirmed by fluorescence spectroscopy. Consequently, the separation could be attributed to the solvophobic association of the PAH molecules with hydrophobic chains of the DOSS surfactant. In another mode of separation, sulfobutylether-beta-cyclodextrin (SB-beta-CD) separated the 16 PAHs on the formation of inclusion complexes with the PAHs, and exhibited different selectivities for the PAHs compared to DOSS. SB-beta-CD and DOSS were then combined in the running buffer to form a mixed pseudo-stationary phase for the separation of the 16 PAHs. Due to the different selectivities of SB-beta-CD and DOSS for the PAHs, the separation of the 16 PAHs was appreciably improved compared to that using DOSS or SB-beta-CD alone. All the 16 PAHs were baseline-resolved using an optimized running buffer containing 22.5 mM DOSS, 15 mM SB-beta-CD, 15% acetonitrile and 5 mM hydroxypropyl-beta-cyclodextrin in 6 mM borate at pH 9.