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Biomedical subjects

Y Feng

Publications and source records attributed to Y Feng.

At least 343 records · Page 19Linked to original sources
PubMed1995

Possible involvement of protein kinases in physical dependence on opioids: studies using protein kinase inhibitors, H-7 and H-8.

Effects of a cAMP-dependent protein kinase and protein kinase C inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) and a cAMP- and cGMP-dependent protein kinase inhibitor, H-8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide), on the behavioral signs of naloxone (an opioid receptor antagonist)-precipitated withdrawal syndrome and effects of H-7 on the change of protein kinase C activity in the pons/medulla region induced by morphine (a mu-opioid receptor agonist) or butorphanol (a mu/delta/kappa mixed opioid receptor agonist) were investigated in this study. Rats were intracerebroventricularly (i.c.v.) infused with morphine (26 nmol/microliters/h) or butorphanol (26 nmol/microliters/h) through osmotic minipumps for 3 days. In some groups, either saline or drug-treated groups were concomitantly infused with H-7 (1 and 10 nmol/microliters/h) or H-8 (10 nmol/microliters/h). The expression of physical dependence produced by morphine or butorphanol, as evaluated by naloxone (5 mg/kg i.p.)-precipitated withdrawal signs, was reduced by concomitant infusion of H-7 or H-8. In the same condition, morphine and butorphanol chronic treatment enhanced (28.1% and 26.3% enhancement over the saline-treated group, respectively) cytosolic protein kinase C activity in the pons/medulla, but not in the membrane fraction. Furthermore, concomitant infusion of H-7 inhibited the enhancement of protein kinase C activity. These results indicate that various types of protein kinases may play an important role in the development and/or expression of physical dependence on opioids. Among them, the enhancement of cytosolic protein kinase C activity in the pons/medulla region seems to be one of the major underlying mechanisms in opioid physical dependence.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

The two pKa's of aspartate-85 and control of thermal isomerization and proton release in the arginine-82 to lysine mutant of bacteriorhodopsin.

To explore the role of Arg82 in the catalysis of proton transfer in bacteriorhodopsin, we replaced Arg82 with Lys, which is also positively charged at neutral pH but has an intrinsic pKa of about 1.7 pH units lower than that of Arg. In the R82K mutant expressed in Halobacterium salinarium, we found the following: (1) The pKa of the purple-to-blue transition at low pH (which reflects the pKa of Asp85) is 3.6 +/- 0.1. At high pH a second inflection in the blue-to-purple transition with pKa = 8.0 is found. The complex titration behavior of Asp85 indicates that the pKa of Asp85 depends on the protonation state of another amino acid residue, X', which has a pKa = 8.0 in R82K. The fit of the experimental data to a model of two interacting residues shows that deprotonation of X' at high pH causes a shift in the pKa of Asp85 from 3.7 to 6.0. In turn, protonation of Asp85 decreases the pKa of X' by 2.3 pH units. This suggests that X' can release a proton upon formation of the M intermediate and the concomitant protonation of Asp85 in the photocycle. (2) The rate constant of dark adaptation, kda, is proportional to the fraction of blue membrane between pH 2 and 10, indicating that thermal isomerization proceeds through the transient protonation of Asp85. The pH dependence of kda shows that two groups with pKal = 3.9 and pKa2 = 8.0 control the rate of dark adaptation in R82K. The 1.7 pH unit shift in pKa2 in R82K compared to the wild type (WT) (pKa2 = 9.7) supports the hypothesis that X' is Arg82 in WT and Lys82 in R82K (or at least that these groups are the principal part of a cluster of residues that constitute X'). (3) Under steady state illumination, the efficiency of proton transport in R82K incorporated in phosphatidylcholine vesicles is at least 40% of that in the WT. A flash-induced transient signal of the pH-sensitive dye pyranine is similar to that in the WT (proton release precedes uptake), but the amplitude is small in R82K (about 15% of that found in the WT), indicating that only a small fraction of protons is released fast in R82K. This supports the suggestions that Arg82 is associated with the proton release pathway (acts as a proton release group or part of a proton release complex) and that Lys cannot efficiently substitute for Arg in this process.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗
PubMed1995

Inhibition and labeling of the coated vesicle V-ATPase by 2-azido-[32P]ATP.

Previous studies have indicated that the 73-kDa A subunit of the coated vesicle V-ATPase possesses a nucleotide-binding site essential for activity (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638) and have identified a cysteine residue (Cys254) whose modification leads to complete loss of activity (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822). To further characterize the structure of the nucleotide-binding sites of the V-ATPase, labeling studies using the photoactivated analog 2-azido-[32P]ATP have been carried out. We have observed that 2-azido-[32P]ATP is hydrolyzed by the V-ATPase at a rate (at 1 mM) approximately 4-fold lower than observed for ATP, indicating that 2-azido-[32P]ATP is a good substrate for the V-ATPase. Irradiation of the V-ATPase in the presence of 0.5 mM 2-azido-[32P]ATP leads to inactivation of V-ATPase activity with a t1/2 of 3-5 min. The 73-kDa A subunit, the 58-kDa B subunit, and the 50-kDa subunit of the AP-2 adaptin complex (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186) are all labeled in an ATP-protectable manner on irradiation of the purified V-ATPase with 2-azido-[32P]ATP. The time course for inactivation most closely correlates with labeling of the A subunit. Measurement of the stoichiometry of 2-azido-[32P]ATP incorporation into the A subunit as a function of inactivation indicates that complete loss of activity is obtained on incorporation of 1.2 mol of 2-azido-[32P]ATP/mol V-ATPase complex. 2-Azido-[32P]ATP labeling indicates that the V-ATPase possesses both rapidly (t1/2 < 2 min) and slowly (t1/2 > 2 min) exchangeable nucleotide-binding sites. The A subunit is labeled upon modification of both rapidly and slowly exchangeable sites whereas the B subunit is labeled upon modification of only rapidly exchangeable sites. Inhibition of V-ATPase activity correlates with labeling of the rapidly exchangeable sites. Amino acid sequence analysis of peptides derived from the 2-azido-[32P]ATP-labeled A subunit indicates labeling of two peptides: a 12-kDa fragment which begins at residue 511 and contains Cys532 and a 3-kDa fragment which begins at residue 233 and contains the glycine-rich loop and Cys254. Only the 12-kDa fragment is labeled upon modification of the rapidly exchangeable sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1995

Ca2+ channel blocker, diltiazem, prevents physical dependence and the enhancement of protein kinase C activity by opioid infusion in rats.

The influence of an L-type Ca2+ channel blocker, diltiazem [(2S-cis)-3-(acetyloxy)-5-[2-(dimethylamino)-ethyl]-2,3-dihydro-2- (4- methoxyphenyl)-1,5-benzothiazepin-4(5H)-one], on the behavioral signs of naloxone (opioid receptor antagonist)-precipitated withdrawal syndrome and the enhancement of protein kinase C activity in the pons/medulla regions of rats rendered dependent on morphine (mu-opioid receptor agonist) or butorphanol (mu/delta/kappa mixed opioid receptor agonist) was investigated. The expression of physical dependence produced by continuous intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/microliters per h) or butorphanol (26 nmol/microliters per h) for 3 days, as evaluated by naloxone (5 mg/kg, i.p.)-precipitated withdrawal signs, was dose dependently attenuated by concomitant infusion of diltiazem (10 and 100 nmol/microliters per h). Furthermore, diltiazem (100 nmol/microliters per h) completely inhibited the enhancement of cytosolic protein kinase C activity in the pons/medulla regions in rats rendered dependent by continuous infusion with morphine or butorphanol. These results suggest that the augmentation of intracellular Ca2+ concentration mediated through L-type Ca2+ channels during continuous opioid infusion leads to the enhancement of cytosolic protein kinase C activity in the pons/medulla region which is intimately involved in the development and/or expression of physical dependence on opioids.

Animals↗
PubMed1995

1H, 13C, and 15N NMR resonance assignments, secondary structure, and backbone topology of a variant of human interleukin-3.

Interleukin-3 (IL-3) is a cytokine which stimulates the proliferation and differentiation of hematopoietic progenitors into multiple cell lineages. The 1H, 15N, and 13C NMR resonances of a recombinant human IL-3 variant (SC-65369) have been assigned using two- and three-dimensional NMR techniques on uniformly 13C/15N-enriched protein. Five helical segments (residues 16-26, 42-50, 55-65, 73-82, and 104-120) and three reverse turns (residues 51-54, 68-71, and 87-90) were identified from the pattern of sequential NOE connectivities, NH(i)-C alpha H(i) scalar coupling constants (3JNH alpha), amide hydrogen exchange data, and the deviation of 13C alpha, 13C beta, 13CO, and C alpha H chemical shifts from random-coil values. Long-range NOEs indicate that the global folding pattern of human IL-3 is a four-helical bundle with an up-up-down-down arrangement of helices that is similar to that of other members of the cytokine family, such as granulocyte-macrophage colony stimulating factor (GM-CSF). A fifth short helix (helix A', residues 42-50) is located in the loop connecting the first and second helices. The absence of helix A' in the corresponding structures of GM-CSF and interleukin-5 suggests that it may be important for recognition of IL-3 by its receptor. The existence of at least two forms of the protein that differ in local conformation was implied from the observation of a limited set of doubled resonances in which each doublet partner had a similar pattern of short-, medium-, and long-range NOEs. The majority of the doubled resonances were close in sequence or space to a proline-rich sequence, which suggested that the different conformational forms of SC-65369 may be caused by slow cis-trans isomerization of proline peptide bonds.

Amino Acid Sequence↗
PubMed1995

Fluorenylalkanoic and benzoic acids as novel inhibitors of cell adhesion processes in leukocytes.

A series of fluoren-9-ylalkanoic and alkylbenzoic acids was prepared as simplified analogues of a previously reported series of antiinflammatory agents which act to inhibit neutrophil recruitment into inflamed tissue. The previous compounds ("leumedins") contained (alkoxycarbonyl)amino or benzoic acid moieties tethered to a fluorene ring. This functionality was replaced with simple structural elements. The new compounds were, in general, found to be more potent than the earlier series at inhibiting adherence of neutrophils to serum-coated wells or endothelial cells in vitro. Compound 9 was approximately 10-fold more potent than the previously reported FMOC-phenylalanine, of which it is an analogue. Similarly, compound 19 was superior in potency to its first generation progenitor, NPC 16570. The new compounds were shown to inhibit neutrophil adherence under conditions in which adherence is mediated by Mac-1 (CD11b/CD18) and LFA-1 (CD11a/CD18); they thus appear to target beta 2-integrins in their antiadhesion activity. These compounds provide a departure point for the further development of new cell adhesion inhibitors which should exhibit enhanced potency and a more selective mode of action.

Benzoates↗
PubMed1995

Translational suppression by trinucleotide repeat expansion at FMR1.

Fragile X syndrome is the result of the unstable expansion of a trinucleotide repeat in the 5'-untranslated region of the FMR1 gene. Fibroblast subclones from a mildly affected patient, each containing stable FMR1 alleles with 57 to 285 CGG repeats, were shown to exhibit normal steady-state levels of FMR1 messenger RNA. However, FMR protein was markedly diminished from transcript with more than 200 repeats. Such transcripts were associated with stalled 40S ribosomal subunits. These results suggest that a structural RNA transition beyond 200 repeats impedes the linear 40S migration along the 5'-untranslated region. This results in translational inhibition by trinucleotide repeat expansion.

Centrifugation, Density Gradient↗
PubMed1995

Influence of chronic opioid treatment on low Km GTPase activity in rat brain: evidence for the involvement of G-proteins in opioid tolerance.

To investigate G protein function during the initial state of opioid tolerance, low Km GTPase activity was measured following chronic treatment with morphine (mu agonist) and butorphanol (mu/delta/kappa mixed agonist) in rats. Chronic opioid administration (20 mg/kg, IP) was performed once a day for 7 consecutive days. Under these conditions, antinociceptive tolerance to morphine but not butorphanol was developed. Chronic morphine treatment enhanced basal low Km GTPase activity in the pons/medulla, but not in the cortex and midbrain. On the other hand, chronic butorphanol treatment had no effect on basal low Km GTPase activity. These results suggest that chronic in vivo treatment of rats with mu agonists leads to an increase in the hydrolysis of GTP to GDP, by a basal low Km GTPase activity of G-proteins in the pons/medulla and that an enhancement of GTPase activity in this specific area may contribute to the development of antinociceptive tolerance to mu agonists.

Animals↗
PubMed1995

Rab 7: an important regulator of late endocytic membrane traffic.

Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.

Animals↗
PubMed1995

[Repair of soft tissue defect on the hand using antebrachial posterior interosseous artery retrograde island flap].

Eight cases of hand defects have been repaired with the antebrachial posterior interosseous artery retrograde island flap. The vessel of the flap is constant and rich in cutaneous branches. The flap has good blood supply, larger surface, appropriate thickness and good skin texture with well-matched color. The operation is simple with high rate of success and does not need to sacrifice a major artery in the forearm.

Adult↗