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Biomedical subjects

X Guo

Publications and source records attributed to X Guo.

At least 19 recordsLinked to original sources
PubMed1995

Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1.

The significance of site-specific phosphorylation of cardiac troponin I (TnI) by protein kinase C and protein kinase A in the regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1 was investigated. The TnI mutants used were T144A, S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase C phosphorylation sites, Thr-144 and Ser-43/ Ser-45, were, respectively, substituted by Ala) and S23A/S24A and N32 (in which the protein kinase A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or deleted). The mutations caused subtle changes in the kinetics of phosphorylation by protein kinase C, and all mutants were maximally phosphorylated to various extents (1.3-2.7 mol of phosphate/mol of protein). Protein kinase C could cross-phosphorylate protein kinase A sites but the reverse essentially could not occur. Compared to wild-type TnI and T144A, un-phosphorylated S43A/S45A, S43A/S45A/T144, S23A/ S24A, and N32 caused a decreased Ca2+ sensitivity of Ca(2+)-stimulated MgATPase of reconstituted actomyosin. S-1. Phosphorylation by protein kinase C of wild-type and all mutants except S43A/S45A and S43A/S45A/T144A caused marked reductions in both the maximal activity of Ca(2+)-stimulated MgATPase and apparent affinity of myosin S-1 for reconstitued (regulated) actin. It was further noted that protein kinase C acted in an additive manner with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a decreased Ca2+ sensitivity of the myofilament. It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca2+ sensitivity of the myofilament, and that phosphorylation of Ser-43/Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca2+ sensitivity, respectively, of actomyosin S-1 MgATPase.

Actomyosin↗
PubMed1995

Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys 84 allowed measurement of the relative distances to the 13C-methyl groups of the 10 methionines of cTnC in the monomer or complex. All 10 1H-13C correlations in the heteronuclear single- and multiple-quantum coherence (HSMQC) spectrum of [13C-methyl] Met cTnC in the complex with cTnI(33-211) were previously assigned [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., & Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735]. In the presence of oxidized spin label, nine of the 10 Met methyl 1H-13C correlations of cTnC were significantly broadened in the cTnC(C35S) monomer. This suggests flexibility within the central helix, or interdomain D/E helical linker, bringing the N- and C-terminal domains in closer proximity than predicted from the crystallographic structure of TnC. In the spin-labeled cTnC(C35S). cTnI(33-211) complex only N-terminal Met methyl 1H-13C correlations of cTnC(C35S) were paramagnetically broadened beyond detection, whereas correlations for Met residues (103, 120, 137, and 157) in the C-terminal domain were not. Thus, complex formation with cTnI decreases interdomain flexibility and maintains cTnC in an extended conformation. This agrees with the recently published study suggesting that sTnC is extended when bound to sTnI [Olah, G. A., & Trewhella, J. (1994) Biochemistry 33, 12800-12806]. The recombinant N-terminal domain of cTnI, cTnI(33-80), gave similar results as observed with cTnI(33-211) when complexed with spin-labeled cTnC(C35S). However, complex formation with the C-terminal fragment, cTnI(86-211), which contains the inhibitory sequence, is insufficient to maintain cTnC extended to the amount observed with either cTnI(33-211) or cTnI(33-80); although compared to that observed in free cTnC, it does cause decreased flexibility in the interdomain linker. In the absence of the N-terminal domain of cTnI, there is a decrease in flexibility within the N-terminal domain of cTnC. Interestingly, the N-terminal domain of cTnC in the reduced spin-labeled complex with cTnI(86-211), in the presence of ascorbate, showed two distinct conformations which were not seen in the complex with cTnI(33-211).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

The effects of nimodipine on the EEG of substance abusers.

Cocaine abusers have increased EEG beta and areas of reduced cortical blood flow. Since, nimodipine has neuroprotective effects and increases blood flow, we investigated the efficacy of single and multiple doses of the nimodipine in normalizing the EEG of substance abusers. Fourteen subjects received single (0, 30, 60 mg) and eleven received multiple daily (up to 150 mg in 12 hours) doses of nimodipine to determine whether this drug would increase EEG alpha and decrease beta in substance abusers. The EEG was recorded from eight scalp locations (F3, C3, P3, O1, F4, C4, P4 and O2) for three minutes during eyes closed, and eyes open conditions. Single and multiple doses of nimodipine produced significant increases in EEG alpha and decreases in EEG beta in the eyes open condition. Thus, nimodipine may have potential therapeutic implications in the treatment of cocaine dependence. Chronic nimodipine dosing in cocaine-dependent individuals is now needed to confirm its efficacy in the treatment of cocaine dependence.

Adult↗
PubMed1995

Nimodipine improves information processing in substance abusers.

We examined whether nimodipine can improve information processing in healthy drug abusers using cognitive event-related potential (ERP) methodology. Placebo and 30- and 60-mg doses of nimodipine were administered on separate days in a random double-blind design to twelve male subjects, who used cocaine and/or opiates as well as alcohol and marijuana. The subjects performed the auditory rare event monitoring (AREM) task and the paired letter version of the visual continuous performance task (CPT) before oral drug administration as well as one and two hours after drug ingestion. The EEG was recorded from 7 scalp locations. The P3 component of the ERPs to the target stimulus was reduced with repeated testing on the placebo day. The 30-mg dose of nimodipine blocked the decrease in P3, which reflects stimulus evaluation in both tasks. Chronic administration of nimodipine may alleviate the cognitive deficits observed in substance abusers during abstinence and prevent treatment relapse.

Adult↗
PubMed1995

Anticancer activity of beta-L-dioxolane-cytidine, a novel nucleoside analogue with the unnatural L configuration.

Naturally occurring nucleosides and all anticancer nucleoside analogue drugs are in the beta-D configuration. L-(-)-dioxolane-cytidine [(-)-OddC] is the first L-nucleoside analogue ever shown to have anticancer activity. This compound was converted within cells to its mono-, di-, and triphosphate metabolites and was incorporated into DNA. As with cytosine arabinoside, conversion to the monophosphate was catalyzed by cellular deoxycytidine kinase, which was essential for cytotoxicity. However, unlike cytosine arabinoside, (-)-OddC was not susceptible to degradation by deoxycytidine deaminase. Because (-)-OddC inhibited the growth of hepatocellular and prostate tumors that are generally difficult to treat, it is a promising candidate for additional testing. Our results indicate that there is a great deal of variability in the chiral specificities of cellular enzymes and demonstrate how these differences can be exploited in the design of better anti-viral and anticancer drugs.

Animals↗
PubMed1995

5-Fluoro-2-pyrimidinone, a liver aldehyde oxidase-activated prodrug of 5-fluorouracil.

5-Fluorouracil (5-FU) is an effective antitumor agent used in treating various cancers. Because of its metabolism by intestinal and other cells, 5-FU has an inconsistent bioavailability that limits its oral use. 5-Fluoro-2-pyrimidione (5-FP), a 5-FU prodrug, was synthesized and found to be converted to 5-FU by aldehyde oxidase, an enzyme present in high concentrations in the livers of mice and humans but not in the gastrointestinal tract. Using BDF1 mice, the pharmacokinetics of 5-FP were studied and compared with those of 5-FU. The bioavailability of 5-FP given orally was 100% at a dosage of 25 mg/kg and 78% at a dosage of 50 mg/kg. The half-lives of both doses of 5-FP were at least 2-fold longer than the half-lives of the same doses of 5-FU, and the clearance rates of 5-FP were 3-fold slower. 5-FP was converted rapidly to 5-FU, in vivo. The resulting 5-FU was measured at a steady-state level of 40-70 microM in plasma, at a dosage of 25 mg/kg, that was sustained for at least 4 hr. Also, when given orally, 5-FP was shown to have potent activity against Colon 38 tumor cells and P388 leukemia cells in mice. The therapeutic index of 5-FP was similar to that of 5-FU in these mouse tumor models. The potential clinical use of 5-FP as a prodrug of 5-FU should be considered.

Administration, Oral↗
PubMed1995

The unique amino-terminal peptide of cardiac troponin I regulates myofibrillar activity only when it is phosphorylated.

Protein kinase A (PKA) dependent phosphorylation of C-protein and cardiac troponin I (cTnI) is known to be associated with a reduced sensitivity to Ca2+. We have investigated the relative importance of each of these sites of phosphorylation in this effect by use of extraction/reconstitution experiments and mutagenesis of recombinant cTnI. Conditions developed for extraction of troponin (Tn) complex also resulted in extraction of C-protein. A truncated cTnI (cTnI/NH2) lacking the 32 amino acids in the unique amino terminal extension of cTnI was engineered and expressed. In contrast to native cTnI, cTnI/NH2, which lacks Ser23 and Ser24, was not phosphorylated by PKA either in pure form or after incorporation into the myofilament lattice. The relation between pCa (-log molar free Ca2+ concentration) and MgATPase activity of non-phosphorylated native myofibrils or non-phosphorylated myofibrils reconstituted with cTnI, but lacking C-protein, was the same and could not be distinguished from that of control or PKA-treated myofibrils into which we exchanged cTnI with cTnI/NH2. However, PKA-dependent phosphorylation of either native myofibrils or reconstituted myofibrils containing cTnI but lacking C-protein resulted in an identical and significant rightward shift of pCa50 (half-maximally activating pCa) in the pCa-activity relationship. Our results strongly indicate that phosphorylation of cTnI at Ser residues in the unique amino terminal extension of the molecule is both necessary and sufficient for the decrease in myofilament Ca(2+)-sensitivity associated with PKA-dependent phosphorylation.

Animals↗
PubMed1995

Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons.

Sympathetic neurons from perinatal rat pups extend only a single axon when maintained in culture in the absence of glia and serum. Exposure to recombinant osteogenic protein-1 (OP-1) selectively induces the formation of dendrites that correctly segregate and modify cytoskeletal and membrane proteins and form synaptic contacts of appropriate polarity. OP-1 requires nerve growth factor (NGF) as a cofactor, and, in the presence of optimal concentrations of NGF, OP-1-induced dendritic growth from cultured perinatal neurons is comparable to that observed in situ. Sympathetic neuroblasts that had not formed dendrites in situ also responded to OP-1 in culture, indicating that OP-1 can cause de novo formation as well as regeneration of dendrites. These data imply that specific signals can regulate the development of neuronal shape and polarity.

Animals↗
PubMed1995

Efficient selection of recombinant adenoviruses by vectors that express beta-galactosidase.

Adenovirus serotype 5 vectors which contain the Excherichia coli beta-galactosidase gene driven by the cytomegalovirus immediate-early promoter as a screenable marker have been made and successfully used in the construction of recombinant adenoviruses. The beta-galactosidase gene has been introduced into viruses in which the E3 region is maintained or deleted and in which the cis-acting packaging sequence has been reiterated at the right end of the chromosome. A unique BstBI site has been introduced 3' of the beta-galactosidase gene. Cotransfection of BstBI-digested vector DNA and a plasmid containing the left end of the viral chromosome followed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) results in clear plaques when overlap recombination has occurred and blue plaques when ligation of the viral arms has occurred within the host cell. The beta-galactosidase-expressing viruses grow to lower titers than do the parental viruses, leading to a relative growth advantage for viruses resulting from overlap recombination. Combined with color selection based on the beta-galactosidase gene, this system permits efficient production and selection of recombinant viruses after cotransfection of BstBI-digested viral DNA with a plasmid including left-end viral sequences and the gene of interest. The beta-galactosidase-expressing viral DNAs were used to construct viruses containing BstBI sites on either side of the cis-acting packaging element as a means of testing their utility.

Adenoviridae↗
PubMed1995

Adenovirus type 5 precursor terminal protein-expressing 293 and HeLa cell lines.

HeLa and 293 cell lines that express biologically active adenovirus type 5 precursor terminal protein (pTP) have been made. The amount of pTP synthesized in these cell lines ranges from barely detectable to greater than that observed in cells infected with the wild-type virus. The pTP-expressing cell lines permit the growth of a temperature-sensitive terminal protein mutant virus sub100r at the nonpermissive temperature. A higher percentage of the stably transfected 293 cell lines expressed terminal protein, and generally at considerably higher levels, than did the HeLa cell lines. While 293 cells appeared to tolerate pTP better than did HeLa cells, high-level pTP expression in 293 cells led to a significantly reduced growth rate. The 293-pTP cell lines produce infectious virus after transfection with purified viral DNA and form plaques when overlaid with Noble agar after infection at low multiplicity. These cell lines offer promise for the production of adenoviruses lacking pTP expression and therefore completely defective for replication.

Adenoviruses, Human↗
PubMed1995

Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.

3T3 Cells↗
PubMed1995

Ca2+ and cAMP activate different K+ conductances in the human intestinal goblet cell line HT29-Cl.16E.

The mechanism of regulated Cl- secretion was evaluated in the mucin-secreting cell line HT29-Cl.16E by transepithelial electrophysiology and fura 2 measurements of cytosolic Ca2+. Carbachol by itself was a weak secretagogue, but augmented adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretion more than twofold, consistent with activation of a rate-limiting K+ conductance. To characterize this conductance, monolayers were apically permeabilized with amphotericin B. At least two types of K+ conductances were identified. One type was activated by elevated cytosolic cAMP levels and inhibited by Ba2+ (inhibitor constant 0.3 mM) in the basolateral solution but was not affected by quinidine or elevated cytosolic Ca2+. The other type was activated by carbachol via cytosolic Ca2+ and was partially inhibited by quinidine (60% inhibition by 2.5 mM quinidine) but was not affected by Ba2+ up to 1 mM. Both conductances appear to be involved in active, transepithelial Cl- secretion in intact monolayers but under different conditions because 1) the cAMP-stimulated short-circuit current (Isc) can be partially inhibited by 1 mM Ba2+ (50%) but not quinidine, 2) the Ba2+ inhibition does not affect the carbachol-induced increase in Isc in cells with elevated cAMP levels, and 3) the carbachol-dependent Isc can be inhibited by quinidine. Therefore, the contribution of the cAMP-dependent K+ conductance appears important for maintaining the membrane potential and therewith Cl- secretion when cAMP is the only messenger for secretion signals, whereas the Ca(2+)-dependent K+ conductance is responsible for the carbachol-stimulated increase in Isc.

Amphotericin B↗
PubMed1995

Hemocyte alterations during melanotic encapsulation of Brugia malayi in the mosquito Armigeres subalbatus.

The involvement of hemocytes in melanotic encapsulation reactions against Brugia malayi was assessed in Armigeres subalbatus. Hemocyte populations, epitope changes, phenol oxidase (PO) activity, and the presence of an 84-kDa polypeptide were investigated in mosquitoes exposed to a B. malayi-infective bloodmeal (= immune-activated), in mosquitoes given a noninfective bloodmeal (= controls), in nonbloodfed mosquitoes (= naive), or in some combination of these. Total hemocyte populations in immune-activated mosquitoes significantly decreased at 24 hr postbloodmeal (PB) as compared with controls. At 48 and 72 hr PB, hemocyte population levels in immune-activated mosquitoes increased to control levels. Epitope changes, as indicated by wheat germ agglutinin (WGA) binding, also were observed. There was a significant increase in the percentage of hemocytes binding WGA in immune-activated mosquitoes at 24 hr PB as compared with controls. Furthermore, the activity of hemocyte PO, an enzyme involved in the melanotic encapsulation pathway, was significantly elevated at 12 hr PB in immune-activated mosquitoes as compared with controls. Analysis for the presence of an 84-kDa polypeptide in A. subalbatus indicates that a 2.0-kb message in total RNA hybridized to D6.12, an Aedes aegypti cDNA encoding an 84-kDa polypeptide that is associated with melanotic encapsulation responses. The hybridization of D6.12 to RNA was not greater in immune-activated as compared to control A. subalbatus, as has been observed in A. aegypti. Results indicate that these hemocyte changes correspond in time with the melanotic encapsulation reactions of A. subalbatus against filarial worms.

Animals↗
PubMed1995

A case-control study of the etiology of laryngeal cancer in Liaoning Province.

A case-control study of laryngeal cancer was conducted in Liaoning Province between January 1991 and April 1992 among 100 subjects with newly diagnosed cancer and 100 controls. The results of one factor analysis, odd ratio (OR), and conditional logistic regression indicated that smoking was the first significant risk factor for laryngeal cancer with an OR value of 16.8. Tobacco smoking ranked the first with an OR value of 30.4. Other factors such as alcoholic indulgence, insufficient intake of vegetable and fruits in winter and spring, air pollution in room and working environment, and fatigue of vocal cords were closely associated with the occurrence of laryngeal cancer.

Carcinoma, Squamous Cell↗
PubMed1995

Effect of hepatocytes from normal or endotoxin treated animals on the production of tumour necrosis factor, interleukin-6, and prostaglandin-E2 by macrophages incubated in vitro with various fatty acids.

OBJECTIVE: To find out if the in vitro production of tumour necrosis factor (TNF), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) by macrophages (P3888D1) were influenced by the addition to the culture media of hepatocytes from normal guinea pigs and from guinea pigs after infusion with endotoxin with and without fatty acids. DESIGN: Laboratory experiment. SETTING: University hospital, United States MATERIAL: 12 male Hartley guinea pigs. MAIN OUTCOME MEASURES: Concentrations of TNF-alpha, IL-6, and PGE2. RESULTS: Hepatocytes inhibited the production of TNF by macrophages. Those from animals pretreated with endotoxin caused a large increase in the production of PGE2 and affected the production of IL-6 only in the presence of fatty acids. Fatty acids differed from each other in their effects on production of TNF-alpha and PGE2, but had no effect on the production of IL-6. CONCLUSION: These results support the concept that hepatocytes and macrophages may interact in vivo, and this interaction may be influenced both by fatty acids that arise from lipid metabolism and from endotoxin present during sepsis and translocation.

Animals↗
PubMed1994

NMR studies delineating spatial relationships within the cardiac troponin I-troponin C complex.

NMR spectroscopy and selective isotope labeling of both recombinant cardiac troponin C (cTnC3) and a truncated cardiac troponin I (cTnI/NH2) lacking the N-terminal 32-amino acid cardiac-specific sequence have been used to probe protein-protein interactions central to muscle contraction. Using [methyl-13C]Met-labeled cTnC3, all 10 cTnC Met residues of Ca(2+)-saturated cTnC3 could be resolved in the two-dimensional heteronuclear single- and multiple-quantum coherence spectrum of the cTnI.cTnC complex. Based on the known Met assignments in cTnC3, the largest chemical shift changes were observed for Met81, Met120, Met137, and Met157. Methionines 120, 137, and 157 are all located in the C-terminal domain of cTnC. Methionine 81 is located at the N terminus of the central helix. Minimal chemical shift changes were observed for Met45, Met47, and Met103 of cTnC3 in the cTnI.cTnC complex. All 6 Met residues in [methyl13C]Met-labeled cTnI/NH2 could be resolved in the cTnI.cTnC complex, suggesting that both cTnI and cTnC form a stable homogeneous binary complex under the conditions of the NMR experiment. In the absence of added protease inhibitors in the cTnI.cTnC complex, cTnI/NH2 was found to undergo selective proteolysis to yield a 5.5-kDa N-terminal fragment corresponding to residues 33-80. Judging from the NMR spectra of [methyl13C]Met-labeled cTnC3, cTnI-(33-80) was sufficient for interaction with the C-terminal domain of cTnC in a manner identical to that observed for native cTnI/NH2. However, in the presence of the proteolytic fragment cTnI-(33-80), the chemical shift of Met81 was not perturbed from its position in free cTnC3. Thus, residues located C-terminal to Arg80 in cTnI appear to be responsible for interaction with the N-terminal half of cTnC. Taken together, these results provide strong evidence for an antiparallel arrangement for the two proteins in the troponin complex such that the N-terminal portion of cTnI interacts with the C-terminal domain of cTnC. This interaction likely plays a role in maintaining the stability of the TnI.TnC complex.

Animals↗