Biomedical subjects

Li Zhang

Publications and source records attributed to Li Zhang.

At least 649 records · Page 36Linked to original sources

PubMed2002

The antioxidant (-)-epigallocatechin-3-gallate inhibits activated hepatic stellate cell growth and suppresses acetaldehyde-induced gene expression.

Activated hepatic stellate cells (HSC) are the primary source of excessive production of extracellular matrix during liver fibrogenesis. Although the underlying mechanisms remain incompletely understood, it is widely accepted that oxidative stress plays a critical role in liver fibrogenesis. Suppression of HSC growth and activation, as well as induction of apoptosis, have been proposed as therapeutic strategies for treatment and prevention of this disease. In the present report, we elucidated, for the first time, effects of the antioxidant (-)-epigallocatechin-3-gallate (EGCG), a major (and the most active) component of green tea extracts, on cultured HSC growth and activation. Our results revealed that EGCG significantly inhibited cultured HSC growth by inducing cell cycle arrest and apoptosis in a dose- and time-dependent manner. In addition, EGCG markedly suppressed the activation of cultured HSC as demonstrated by blocking transforming growth factor-beta signal transduction and by inhibiting the expression of alpha1(I) collagen, fibronectin and alpha-smooth muscle actin genes induced by acetaldehyde, the most active metabolite of ethanol. Furthermore, EGCG reacted differently in the inhibition of nuclear factor-kappaB activity between cultured HSC with or without acetaldehyde stimulation. Taken together, our results indicated that EGCG was a novel and effective inhibitor for activated HSC growth and activation in vitro. Further studies are necessary to evaluate the effect of this polyphenol in prevention of quiescent HSC activation in vivo, and to further elucidate the underlying mechanisms.

Alcohols↗

PubMed2002

Nonoxidizable ubiquinol derivatives that are suitable for the study of the ubiquinol oxidation site in the cytochrome bc1 complex.

Recent X-ray crystallographic analyses of the mitochondrial cytochrome bc1 complex show ubiquinone binding at the Q(i) site, but attempts to show binding of ubiquinol or ubiquinone at the Q(o) site have been unsuccessful, even though the binding of noncompetitive Q(o) site inhibitors near the putative ubiquinol binding pocket is well established. We speculate that ubiquinol binds transiently to the Q(o) site only when both heme b(L) and the iron sulfur cluster are in the oxidized form, an experimental condition difficult to obtain since ubiquinol will be oxidized once bound to the site. Stable binding at the Q(o) site might be achieved by a nonoxidizable ubiquinol-like compound. For this purpose, the isomers 2,3,4-trimethoxy-5-decyl-6-methyl-phenol (TMDMP) and 2,3,4-trimethoxy-5-methyl-6-decyl-phenol (TMMDP) were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1, 4-benzoquinol (Q0C10) by controlled methylation and separated by TLC and HPLC. The structures of TMDMP and TMMDP were established by 1H-13C-two-dimensional NMR. Both are competitive inhibitors of the cytochrome bc1 complex, with TMDMP being the stronger one. Preliminary results suggest that TMDMP binds tightly enough to make X-ray crystallography of inhibitor-bc1 complex co-crystals feasible. The binding site of TMDMP does not overlap with the binding sites of stigmatellin, MOA-stilbene (MOAS), undecylhydroxydioxobenzothiazole (UHDBT) and myxothaizol.

Animals↗

PubMed2002

Endogenous cannabinoid, anandamide, acts as a noncompetitive inhibitor on 5-HT3 receptor-mediated responses in Xenopus oocytes.

The cloned 5-HT3 receptor from NCB-20 neuroblastoma cells was expressed in Xenopus oocytes and the effect of the endogenous cannabinoid ligand, anandamide, was investigated on the function of this receptor. The oocytes expressing the cloned 5-HT3 receptors were voltage-clamped at -70 mV. Anandamide, at the concentration range of 0.1-100 microM, reversibly inhibited 1 microM 5-HT induced currents. The inhibition of 5-HT induced currents by anandamide was concentration-dependent with an EC50 of 3.7 microM and slope value of 0.94. This inhibitory effect was not dependent on the membrane potential and anandamide did not have an effect on the reversal potential of 5-HT-induced currents. In the presence of 10 microM anandamide, the maximum 5-HT-induced response was also inhibited and the respective EC50 values were 3.4 microM and 3.1 microM in the absence and presence of anandamide, indicating that anandamide acts as a noncompetitive antagonist on 5-HT3 receptors. CB1 receptor antagonist SR-141716A (1 microM) and pertussis toxin (5 microg/ml) did not cause a significant change on the inhibition of 5-HT responses by anandamide. The effect of anandamide was not changed by preincubating the oocytes with 0.2 mM 8-Br-cAMP, a membrane-permeable analog of cAMP, or Sp-cAMPS (0.1 mM), a membrane-permeable protein kinase A activator. These results suggest that the effect of anandamide is independent of the activation of cAMP pathway and not mediated by the activation of PTX sensitive G-proteins. In conclusion, we demonstrated that the endogenous cannabinoid anandamide inhibits the function of 5-HT3 receptors expressed in Xenopus oocytes in a cannabinoid-receptor independent and noncompetitive manner.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2002

A new look at calretinin-immunoreactive amacrine cell types in the monkey retina.

We have examined amacrine cells that are calretinin-immunoreactive (-IR) in the macaque monkey retina with the aim of classifying them into morphological and functional subtypes. There are calretinin-IR cells in the fovea and throughout the retina. Their highest density is reached at 1.0 mm from the foveal pit (10500 cells/mm(2)) and falls to 2600/mm(2) by 10 mm of eccentricity. Nearest-neighbor statistics for the calretinin-IR cell body distribution indicate a nonregular pattern, with a regularity index of 1.4-1.6. There is an increase or "bump" of cell density 3.5-4.0 mm from the foveal pit, corresponding to the rod photoreceptor density peak. Based on morphological differences, there appear to be three types of amacrine cell that are calretinin-IR. To determine the types, we doubly immunolabeled retinas, from fovea to periphery, for calretinin-IR in combination with other calcium binding proteins and inhibitory amino acid neurotransmitters. Labeling with parvalbumin and calretinin antibodies indicated that 70% of the amacrine cells were solely calretinin-IR, and 30% contained parvalbumin-IR as well. In the same way, 70% of the calretinin-IR amacrine cells colocalized calbindin, but 30% were only calretinin-IR. Among the calretinin/calbindin-colocalized cells, there were small-field and wide-field types. Double labeling with antibodies to calretinin and gamma-aminobutyric acid (GABA) and to calretinin and glycine revealed the majority to be glycine-IR, but some were GABA-IR. The glycine-IR population consists mainly of AII amacrine cell types, but clearly another non-AII type is involved. The non-AII glycine-IR population resembles a small- to medium-field diffuse type. The calretinin-IR wide-field type is GABAergic and corresponds to an A19 type. The central, rod-free, fovea contains the calretinin-IR, non-AII glycine-IR type and the calretinin-IR, GABAergic type only. To learn more concerning the circuitry of the calretinin/glycine-IR, non-AII amacrine cell type in isolation from AII amacrine cells, we concentrated on the rod-free fovea, where AII amacrine cells are absent. We performed a serial section electron microscopy (EM) study on four calretinin-IR cells. They were involved with cone pathway circuitry. They got input from ON and OFF midget bipolar cells, reciprocated synapses to these bipolar cells, and provided synapses to ON-center ganglion cells. Thus we have obtained new information on a cone pathway amacrine cell of the central monkey fovea that is involved in the midget system.

Amacrine Cells↗

PubMed2002

The closed state of a H+ channel helical bundle combining precise orientational and distance restraints from solid state NMR.

An interhelical distance has been precisely measured by REDOR solid-state NMR spectroscopy in the transmembrane tetrameric bundle of M2-TMP, from the M2 proton channel of the influenza A viral coat. The high-resolution structure of the helical backbone has been determined using orientational restraints from uniformly aligned peptide preparations in hydrated dimyristoylphosphatidylcholine bilayers. Here, the distance between (15)N(pi) labeled His37 and (13)C(gamma) labeled Trp41 is determined to be less than 3.9 A. Such a short distance, in combination with the known tilt and rotational orientation of the individual helices, permits not only a determination of which specific side chain pairings give rise to the interaction, but also the side chain torsion angles and restraints for the tetrameric bundle can also be characterized. The resulting proton channel structure is validated in a variety of ways. Both histidine and tryptophan side chains are oriented in toward the pore where they can play a significant functional role. The channel appears to be closed by the proximity of the four indoles consistent with electrophysiology and mutagenesis studies of the intact protein at pH 7.0 and above. The pore maintains its integrity to the N terminal side of the membrane, and at the same time, a cavity is generated that appears adequate for binding amantadine. Finally, the observation of a 2 kHz coupling in the PISEMA spectrum of (15)N(pi)His37 validates the orientation of the His37 side chain based on the observed REDOR distance.

Amino Acid Sequence↗

PubMed2002

Donor-lymphocyte infusion induces transplantation tolerance by activating systemic and graft-infiltrating double-negative regulatory T cells.

Donor-lymphocyte infusion (DLI) before transplantation can lead to specific tolerance to allografts in mice, nonhuman primates, and humans. We and others have demonstrated a role for regulatory T cells in DLI-induced, donor-specific transplantation tolerance, but it is not known how regulatory T cells are activated and where they execute their function. In this study, we observed, in both transgenic and normal mice, that DLI before transplantation is required for activation of alphabeta-T-cell-receptor-positive, CD3(+)CD4(-)CD8(-) double-negative (DN) regulatory T cells in the periphery of recipient mice. More interestingly, DLI induced DN regulatory T cells to migrate preferentially to donor-specific allogeneic skin grafts and to form a majority of graft-infiltrating T cells in accepted skin allografts. Furthermore, both recipient-derived peripheral and graft-infiltrating DN T cells were able to suppress and kill antidonor CD8(+) T cells in an antigen-specific manner. These data indicate that DLI may induce donor-specific transplantation tolerance by activating recipient DN regulatory T cells in the periphery and by promoting migration of regulatory T cells to donor-specific allogeneic skin grafts. Our results also show that DN regulatory T cells can eliminate antidonor T cells both systemically and locally, a finding suggesting that graft-infiltrating T cells can be beneficial to graft survival.

Animals↗

PubMed2002

Distinct molecular basis for differential sensitivity of the serotonin type 3A receptor to ethanol in the absence and presence of agonist.

Ethanol can potentiate serotonin type 3 (5-HT(3)) receptor-mediated responses in various neurons and in cells expressing 5-HT(3A) receptors. However, the molecular basis for alcohol modulation of 5-HT(3) receptor function has not been determined. Here we report that point mutations of the arginine at amino acid 222 in the N-terminal domain of the 5-HT(3A) receptor can alter the EC(50) value of the 5-HT concentration-response curve. Some point mutations at amino acid 222 resulted in spontaneous opening of the 5-HT(3A) receptor channel and an inward current activated by ethanol in the absence of agonist. Among these mutant receptors, the amplitude of the current activated by ethanol in the absence of agonist was correlated with the amplitude of the current resulting from spontaneous channel openings, suggesting that the sensitivity of the receptor to ethanol in the absence of agonist is, at least in part, dependent on the preexisting conformational equilibrium of the receptor protein. On the other hand, point mutations that conferred greater sensitivity to ethanol potentiation of agonist-activated responses were less sensitive or insensitive to ethanol in the absence of agonist. For these receptors, the magnitude of the potentiation of agonist-activated responses by ethanol was inversely correlated with the EC(50) values of the 5-HT concentration-response curves, suggesting that these mutations may modulate ethanol sensitivity of the receptor by altering the EC(50) value of the receptor. Thus, distinct molecular processes may determine the sensitivity of 5-HT(3A) receptors to ethanol in the absence and presence of agonist.

Amino Acid Sequence↗

PubMed2002

The crystal structure of mitochondrial cytochrome bc1 in complex with famoxadone: the role of aromatic-aromatic interaction in inhibition.

Ubiquinol cytochrome c oxido-reductase (EC. 1.10.2.2, bc1) is an integral membrane protein complex essential to cellular respiration. Structures of the 11-subunit mitochondrial bc1 complex were determined with and without the fungicide famoxadone. Specific inhibition by famoxadone is achieved through a coordinated optimization of aromatic-aromatic interactions where conformational rearrangements in famoxadone and in residues lining the inhibitor-binding pocket produce a network of aromatic-aromatic interactions that mimic the crystal lattice of benzene. The profound aromatic-aromatic interactions as supported by prior mutagenesis provide a structural basis for specific protein-ligand interaction in a hydrophobic environment. Dramatic conformational changes, both in cyt. b and ISP subunits in the inhibitor-protein complex, confer experimental evidence for a functional role of cytochrome b in the induced conformational arrest of ISP and allow the identification of a possible intrasubunit signal transduction pathway that controls the movement of ISP. These results support an inhibitory mechanism that is consistent with the requirement for ISP movement in the electron transfer of this complex.

Amino Acid Sequence↗

PubMed2002

Identification of functional segments within the beta2I-domain of integrin alphaMbeta2.

The alpha(M)beta(2) integrin plays an important role in leukocyte biology through its interactions with a diverse set of ligands. Efficient ligand binding requires the involvement of both the alpha(M) and beta(2) subunits. Past ligand binding studies have focused mainly on the alpha(M) subunit, with the beta(2) subunit being largely unexplored. Therefore, in this study we conducted homolog-scanning mutagenesis on the I-domain (residues 125-385) within the beta(2) subunit. We identified four noncontiguous sequences (Arg(144)-Lys(148), Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309)) that are critical for fibrinogen and C3bi binding to alpha(M)beta(2). Molecular modeling revealed that these four sequences reside within a narrow region on the surface of the beta(2)I-domain, in close proximity to three potential cation-binding sites. Among these sequences, Gln(199)-Ala(203), Leu(225)-Leu(230), and Gly(305)-His(309) are important for the binding of both ligands, whereas Arg(144)-Lys(148) is more critical for fibrinogen than for C3bi binding. These sequences within the beta(2)I-domain are directly involved in ligand binding, since 1) switching these segments to their corresponding beta(1) sequences destroyed ligand binding; 2) loss of function was not due to a nonspecific gross conformational change, since the defective alpha(M)beta(2) mutants reacted well with a panel of conformation-dependent mAbs; 3) mutation of these functional sequences did not effect Ca(2+) binding; and 4) synthetic peptides corresponding to sequences Gln(199)-Ala(203) and Gly(305)-His(309) blocked ligand binding to alpha(M)beta(2), and the peptides interacted directly with fibrinogen and C3bi. Given the similarity among all integrin beta subunits, our results may help us to understand the underlying mechanism of integrin-ligand interactions in general.

Amino Acid Sequence↗

PubMed2002

[Influence of high-frequency electric surgical knife on healing of abdominal incision, experimental and clinical studies].

OBJECTIVE: To study the influence of high-frequency electric surgical knife on abdominal incision healing. METHODS: Two hundred and forty Wistar rats were randomly divided into two groups of 120 rats to undergo abdominal incision by high-frequency electric knife or common lancet respectively. Each of these two groups was redivided into four subgroups that were injected hypodermically with 0.2 ml of quantitative mixture of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at the concentrations of 0.24 x 10(2) bacteria (10(2) group), 4.49 x 10(4) bacteria (10(5) group), and 4.11 x 10(7) bacteria (10(8) group) respectively, and normal saline of the same volume (10(0) group). Eight days after the operation, the rats were killed. The infection rate of the operational wound was observed and the tissues around the wounds were examined pathologically. On the basis of the animal experiment, 220 patients undergoing abdominal operation above type II were randomly allocated into one of following three groups: high-frequency electric knife (EK) group (93 cases, high-frequency electric knife was used to cut the abdominal tissues and electro-coagulation was used for hemostasis), electro-coagulation (EC) group (55 cases, the abdominal tissues were cut with common lancet and electro-coagulation was used for hemostasis) and control group (72 cases, common lancet and silk thread suture were used). The healing of wound was observed after operation. RESULTS: Four rats died of anesthetic accident; the other 236 rats were killed 8 days after. The wound infection rates were 23.33% and 6.60% in the experimental 10(0) group and the control 10(0) group respectively (chi(2) = 3.28, P > 0.05); 37.04% and 13.33% in the experimental 10(2) group and the control 10(2) group respectively (chi(2) = 4.31, P > 0.05), 50.00% and 24.14% in the experimental 10(5) group and the control 10(5) group respectively (chi(2) = 4.22, P > 0.05); and 63.33% and 36.67% in the experimental 10(8) group and the control 10(8) group respectively (chi(2) = 4.27, P > 0.05). Clinical observation showed a delayed wound healing rate of 17.20% (16 cases) in EK group, 16.36% (11 cases) in EC group, and 2.86% (2 cases) in the control group. There was a statistically significant difference in delayed wound healing rate between the EK and control groups (chi(2) = 8.57, P < 0.01) and between the EC and control groups (chi(2) = 5.66, P < 0.05). However, no significant difference in the delayed wound healing rate was seen between the EK and EC groups (chi(2) = 0.017, P > 0.05). CONCLUSION: High-frequency electric knife remarkably delays the healing of abdominal incision. Its application should be minimized so as to reduce the possibility of postoperative complications.

Abdomen↗

PubMed2002

Direct noncompetitive inhibition of 5-HT(3) receptor-mediated responses by forskolin and steroids.

5-HT(3) receptors cloned from NCB-20 cells were expressed in Xenopus oocytes, and the effects of forskolin and steroids on the function of the receptors were investigated using the two-electrode voltage-clamp technique. Forskolin, 17-beta-estradiol, and progesterone inhibited the currents activated by 1 microM 5-HT in a reversible and concentration-dependent manner, with IC(50) values of 12, 33, and 89 microM, respectively. The inhibitory effects of forskolin and 17-beta-estradiol were independent of the membrane potential. Forskolin and 17-beta-estradiol significantly reduced the maximal amplitude of the 5-HT concentration-response curve (E(max)) without significantly affecting the EC(50), indicating that these compounds act as noncompetitive inhibitors of the 5-HT(3) receptor. The cAMP analogue, 8-Br-cAMP (0.2 mM), and the protein kinase A activator, Sp-cAMP (0.1 mM), did not affect the amplitude of 5-HT(3) receptor-mediated currents. The membrane-permeable protein kinase A inhibitor Rp-cAMP (0.1 mM) and the estrogen-receptor antagonist tamoxifen (1 microM) did not affect the inhibition of 5-HT-activated current. In addition, 5-HT(3) receptor-mediated currents were inhibited by both 1,9-dideoxy forskolin (30 microM), which does not activate adenylyl cyclase, and wForskolin (30 microM), a charged hydrophilic analogue of forskolin that is membrane impermeable. These results indicate that both forskolin and 17-beta-estradiol inhibit the function of the 5-HT(3) receptor in a noncompetitive manner and that this inhibition is independent of cAMP levels.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2002

ATP-sensitive potassium channels participate in glucose uptake in skeletal muscle and adipose tissue.

ATP-sensitive potassium (K(ATP)) channels are known to be critical in the control of both insulin and glucagon secretion, the major hormones in the maintenance of glucose homeostasis. The involvement of K(ATP) channels in glucose uptake in the target tissues of insulin, however, is not known. We show here that Kir6.2(-/-) mice lacking Kir6.2, the pore-forming subunit of these channels, have no K(ATP) channel activity in their skeletal muscles. A 2-deoxy-[(3)H]glucose uptake experiment in vivo showed that the basal and insulin-stimulated glucose uptake in skeletal muscles and adipose tissues of Kir6.2(-/-) mice is enhanced compared with that in wild-type (WT) mice. In addition, in vitro measurement of glucose uptake indicates that disruption of the channel increases the basal glucose uptake in Kir6.2(-/-) extensor digitorum longus and the insulin-stimulated glucose uptake in Kir6.2(-/-) soleus muscle. In contrast, glucose uptake in adipose tissue, measured in vitro, was similar in Kir6.2(-/-) and WT mice, suggesting that the increase in glucose uptake in Kir6.2(-/-) adipocytes is mediated by altered extracellular hormonal or neuronal signals altered by disruption of the K(ATP) channels.

Adenosine Triphosphate↗

PubMed2002

Augmentation of NO-mediated vasodilation in metabolic acidosis.

Reduction of perivascular pH in acidemia produces hyporesponsiveness of vascular bed to vasoconstrictors. In the present study, we examined the effects of modest acidification on dilatory responses of isolated rat thoracic aorta. Acetylcholine produced endothelium-dependent relaxation in phenylephrine-precontracted aorta, which was markedly enhanced by acidification of Krebs-Henseleit solution from pH 7.4 to 7.0. A similar augmentation was observed in the relaxing responses to NO donors (SNP, SIN-1, SNAP), 8-Br-cGMP and NS-1619 (a putative K(Ca) channel opener and/or Ca channel inhibitor) in endothelium-denuded, phenylephrine-contracted aorta. However, papaverine-induced relaxation was not affected by the change in pH. At pH 7.4, the relaxing responses to acetylcholine and SNP were partially inhibited by charybdotoxin (K(Ca) channel inhibitor) but not glibenclamide (K(ATP) channel inhibitor), while at pH 7.0 the relaxation induced by either drug was not affected by K(+) channel inhibitors. Relaxation induced by 8-Br-cGMP or NS-1619 was not inhibited by charybdotoxin or glibenclamide. Acidification to pH 7.0 increased the cGMP production in response to acetylcholine in endothelium-intact aorta and to SNP in endothelium-denuded aorta. These results show that modest acidification augments NO-mediated relaxation in rat aorta, probably due to an enhancement of cGMP-dependent but K(+) channel-unrelated relaxation mechanisms.

Acetylcholine↗

PubMed2002

Adjuvant treatment with neuroserpin increases the therapeutic window for tissue-type plasminogen activator administration in a rat model of embolic stroke.

BACKGROUND: After stroke, the thrombolytic effect of tissue-type plasminogen activator (tPA) in the intravascular space is beneficial, whereas its extravascular effect on ischemic neurons is deleterious. We tested the hypothesis that neuroserpin, a natural inhibitor of tPA, reduces tPA-induced neuronal toxicity and increases its therapeutic window for treatment of embolic stroke. METHODS AND RESULTS: Rats were subjected to embolic middle cerebral artery occlusion (MCAO). Ischemic brains were treated with neuroserpin in combination with recombinant human tPA (n=7), tPA alone (n=7), or saline (n=9). Neuroserpin (20 micro L of 16 micro mol/L active neuroserpin) was intracisternally injected 3 hours and tPA (10 mg/kg) was intravenously administered 4 hours after ischemia. MRI measurements were performed to study blood brain barrier (BBB) leakage and ischemic lesion volume. Administration of tPA alone 4 hours after ischemia significantly (P<0.05) increased BBB leakage in the ischemic core measured by Gd-DTPA-enhanced MRI compared with rats treated with saline. However, treatment with neuroserpin in combination with tPA significantly (P<0.05) reduced BBB leakage, brain edema, and ischemic lesion volume compared with rats treated with tPA alone, although ischemic lesion volumes were the same in both groups before the treatment. Immunostaining revealed that MCAO resulted in reduction of neuroserpin immunoreactivity in the ipsilateral hemisphere after 2 to 6 hours of ischemia. Zymographic assay showed increased plasminogen activity in areas with BBB leakage in rats treated with tPA. CONCLUSIONS: Administration of neuroserpin after stroke is neuroprotective, seemingly because it blocks the extravascular effect of tPA, leading to subsequent decrease in stroke volume and widening of the therapeutic window for the thrombolytic effect of tPA.

Adjuvants, Pharmaceutic↗

PubMed2002

The amino-terminal domain of the E subunit of vacuolar H(+)-ATPase (V-ATPase) interacts with the H subunit and is required for V-ATPase function.

Vacuolar H(+)-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F(1)F(0)-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A(1)-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.

Adenosine Triphosphate↗

PubMed2002

The immune regulatory function of lymphoproliferative double negative T cells in vitro and in vivo.

Lymphoproliferative (lpr) mice, which lack functional Fas receptor expression and develop autoimmune lymphoproliferative disease, have an accumulation of T cell receptor-alphabeta(+)CD4(-)CD8(-) (double negative T cells [DNTC]) in the periphery. The function of the accumulating DNTC is not clear. In this study we demonstrate that B6/lpr DNTC can dose dependently kill syngeneic CD8(+) and CD4(+) T cells from wild-type B6 mice through Fas/Fas ligand interactions in vitro. We also demonstrate that B6/lpr DNTC that are activated and expand in vivo are able to specifically down-regulate allogeneic immune responses mediated by syngeneic Fas(+)CD4(+) and CD8(+) T cells in vivo. B6/lpr DNTC that have been preactivated in vivo by infusion of either class I- (bm1) or class II- (bm12) mismatched allogeneic lymphocytes are able to specifically enhance the survival of bm1 or bm12, but not third-party skin allografts when adoptively transferred into naive B6(+/+) mice. These findings clearly demonstrate that B6/lpr DNTC have a potent immune regulatory function in vitro and in vivo. They also provide new insights into the mechanisms involved in the development of autoimmune disease in lpr mice.

Adoptive Transfer↗

PubMed2002

A nonredundant role for the adapter protein Shc in thymic T cell development.

Signaling via the pre-T cell receptor (pre-TCR) regulates survival, proliferation, allelic exclusion and differentiation of thymocytes. The role played by the adapter protein Shc in T cells has remained controversial, and its role in pre-TCR signaling has not been addressed. We examined Shc function in thymic T cell development using two genetic approaches. Cre-loxP-mediated inducible expression in transgenic mice of a phosphorylation-defective mutant of Shc impaired signaling through the pre-TCR as well as subsequent proliferation and differentiation. Conditional deletion of the Shc locus in thymocytes also affected thymic maturation at the same pre-TCR developmental stage. Thus, both Shc expression and its tyrosine phosphorylation play an essential and nonredundant role in thymic T cell development.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The B' helix determines cytochrome P450nor specificity for the electron donors NADH and NADPH.

Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is unique because it is catalytically self-sufficient, receiving electrons directly from NADH or NADPH. However, little is known about the direct binding of NADH to cytochrome. Here, we report that oxidized pyridine nucleotides (NAD(+) and NADP(+)) and an analogue induce a spectral perturbation in bound heme when mixed with P450nor. The P450nor isoforms are classified according to electron donor specificity for NADH or NADPH. One type (Fnor, a P450nor of Fusarium oxysporum) utilizes only NADH. We found that NAD(+) induced a type I spectral change in Fnor, whereas NADP(+) induced a reverse type I spectral change, although the K(d) values for both were comparable. In contrast, NADP(+) as well as NAD(+) caused a type I spectral change in Tnor, a P450nor isozyme from Trichosporon cutaneum that utilizes both NADH and NADPH as electron donors. The B' helix region of Tnor ((73)SAGGKAAA(80)) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor ((73)SASGKQAA(80)). A single mutation (S75G) significantly improved the NADPH- dependent Nor activity of Fnor, and the overall activity was accelerated via the NADPH-enhanced reduction step. These results showed that pyridine nucleotide cofactors can bind P450nor and that only a few residues in the B' helix region determine cofactor specificity. We further showed that a poor electron donor (NADPH) could also bind Fnor, but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser(75) against the 2'-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding.

Binding Sites↗