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Biomedical subjects

J Tang

Publications and source records attributed to J Tang.

At least 55 records · Page 3Linked to original sources
PubMed2002

Haemoglobin and ATP levels in CSF from a dog model of vasospasm.

Haemoglobin and adenosine 5'-triposphate (ATP) released from lysed erythrocytes have been postulated to be responsible for delayed cerebral vasospasm after subarachnoid haemorrhage (SAH). However, the concentrations of haemoglobin and ATP in cerebrospinal fluid (CSF) in patients or in an animal model of vasospasm have not been reported. In this study, 12 mongrel dogs underwent a double blood injection via the cisterna magna on day 0 and 2, after an initial collection of CSF. On day 3, 5 or 7, the dogs were sacrificed after a second collection of CSF. An angiogram was recorded on day 0 and on the day of sacrifice. Results showed that the diameter of the dog's basilar artery was reduced 20% on day 3 (P > 0.05), 35% on day 5 (P < 0.05) and 45% on day 7 (P < 0.05). The concentrations of OxyHb, deOxyHb and MetHb in CSF were increased (P < 0.05), and all peaked on day 3. OxyHb and MetHb remained significantly higher than control (day 0) from day 3 to day 7, while deOxyHb remained at a high level on day 5 but returned to normal on day 7. In contrast, ATP was decreased (P < 0.05) on days 5 and 7 after SAH compared with day 0. The results indicate that haemoglobin might be involved in the development of cerebral vasospasm. The possible role of ATP in vasospasm remains unclear.

Adenosine Triphosphate↗
PubMed2002

Effects of deletion of streptokinase residues 48-59 on plasminogen activation.

Streptokinase (SK) is a thrombolytic agent widely used for the clinical treatment of clotting disorders such as heart attack. The treatment is based on the ability of SK to bind plasminogen (Pg) or plasmin (Pm), forming complexes that proteolytically activate other Pg molecules to Pm, which carries out fibrinolysis. SK contains three major domains. The N-terminal domain, SKalpha, provides the complex with substrate recognition towards Pg. SKalpha contains a unique mobile loop, residues 45-70, absent in the corresponding domains of other bacterial Pg activators. To study the roles of this loop, we deleted 12 residues in this loop in both full-length SK and the SKalpha fragment. Kinetic data indicate that this loop participates in the recognition of substrate Pg, but does not function in the active site formation in the activator complex. Two crystal structures of the deletion mutant of SKalpha (SKalpha(delta)) complexed with the protease domain of Pg were determined. While the structure of SKalpha(delta) is essentially the same as this domain in full-length SK, the mode of SK-Pg interaction was however different from a previously observed structure. Even though mutagenesis studies indicated that the current complex represents a minor interacting form in solution, the binding to SKalpha(delta) triggered similar conformational changes in the Pg active site in both crystal forms.

Base Sequence↗
PubMed2001

Electrochemistry and spectroscopy study on the interaction of microperoxidase-11 with lipid membrane.

The interaction of microperoxidase-11 (MP11) with cationic lipid vesicles of didodecyldimethylammonium bromide (DDAB) induces an alpha-helical conformation from random coil conformations in solution and this change then makes heme macrocycle more distorted. DDAB-induced MP11 conformations were investigated by cyclic votammetry (CV), circular dichroism (CD) and UV-vis spectrometry. All results indicate that the binding of MP11 in solution to DDAB vesicles and the ordered structure formation are driven by mostly electrostatic interaction between negatively charged residues in the undecapeptide and positively charged lipid headgroups on the membrane surface. Upon binding to DDAB, its half-peak potential was also changed. The mechanism of the interaction between MP11 and DDAB was also discussed.

Circular Dichroism↗
PubMed2001

Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.

Binding Sites↗
PubMed2001

Potentiated amygdaloid auditory-evoked potentials and freezing behavior after fear conditioning in mice.

Elucidation of cellular and molecular mechanisms underlying fear-related memory would greatly benefit from the possibility of combined behavioral and electrophysiological recordings in genetically modified mice. As a first step to this goal, we implanted adult C57BL/6J mice with recording electrodes aimed at the basolateral amygdaloid complex and trained them in an auditory fear conditioning paradigm. After conditioning, animals with paired tone and footshock presentation showed not only intensified freezing behavior lasting for 2 days, but also increases, lasting 4 days, in slope and amplitude of the most negative component of auditory-evoked potentials triggered by the conditioned stimulus. These effects could not be observed in animals with unpaired tone and footshock presentation. Thus, our data show that a long-lasting association of a former neutral tone with an aversive situation is accompanied by a long-lasting increase of auditory-evoked potentials in freely moving mice. However, extinction of the potentiation of auditory-evoked potentials and freezing behavior followed different time courses, thus making a direct relationship between these responses unlikely.

Acoustic Stimulation↗
PubMed2001

Structure-based design: potent inhibitors of human brain memapsin 2 (beta-secretase).

Memapsin 2 (beta-secretase) is one of two proteases that cleave the beta-amyloid precursor protein (APP) to produce the 40-42 residue amyloid-beta peptide (Abeta) in the human brain, a key event in the progression of Alzheimer's disease. On the basis of the X-ray crystal structure of our lead inhibitor (2, OM99-2 with eight residues) bound to memapsin, we have reduced the molecular weight and designed potent memapsin inhibitors. Structure-based design and preliminary structure-activity studies have been presented.

Amyloid Precursor Protein Secretases↗
PubMed2001

Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design.

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].

Alzheimer Disease↗
PubMed2001

A stoichiometric complex of neurexins and dystroglycan in brain.

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.

Alternative Splicing↗
PubMed2001

The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca(2+)-dependent actin binding.

The three-dimensional structure of alpha-actinin from rabbit skeletal muscle was determined by cryoelectron microscopy in combination with homology modeling of the separate domain structures based on results previously determined by X-ray crystallography and nuclear magnetic resonance spectroscopy. alpha-Actinin was induced to form two-dimensional arrays on a positively charged lipid monolayer and micrographs were collected from unstained, frozen hydrated specimens at tilt angles from 0 degrees to 60 degrees. Interpretation of the 15 A-resolution three-dimensional structure was done by manually docking homologous models of the three key domains, actin-binding, three-helix motif and the C-terminal calmodulin-like domains. The initial model was refined quantitatively to improve its fit to the experimental reconstruction. The molecular model of alpha-actinin provides the first view of the overall structure of a complete actin cross-linking protein. The structure is characterized by close proximity of the C-terminal, calmodulin-like domain to the linker between the two calponin-homology domains that comprise the actin-binding domain. This location suggests a hypothesis to explain the involvement of the C-terminal domain in Ca(2+)-dependent actin binding of non-muscle isoforms.

Actinin↗
PubMed2001

Molecular cloning and characterization of chemokine-like factor 1 (CKLF1), a novel human cytokine with unique structure and potential chemotactic activity.

Cytokines are small proteins that have an essential role in the immune and inflammatory responses. The repertoire of cytokines is becoming diverse and expanding. Here we report the identification and characterization of a novel cytokine designated as chemokine-like factor 1 (CKLF1). The full-length cDNA of CKLF1 is 530 bp long and a single open reading frame encoding 99 amino acid residues. CKLF1 bears no significant similarity to any other known cytokine in its amino acid sequence. Expression of CKLF1 can be partly inhibited by interleukin 10 in PHA-stimulated U937 cells. Recombinant CKLF1 is a potent chemoattractant for neutrophils, monocytes and lymphocytes; moreover, it can stimulate the proliferation of murine skeletal muscle cells. These results suggest that CKLF1 might have important roles in inflammation and in the regeneration of skeletal muscle.

Amino Acid Sequence↗
PubMed2001

Structure of the P700(+ )A1(-) radical pair intermediate in photosystem I by high time resolution multifrequency electron paramagnetic resonance: analysis of quantum beat oscillations.

The geometry of the secondary radical pair P700(+)A1(-), in photosystem I (PSI) from the deuterated and 15N-substituted cyanobacterium Synechococcus lividus, has been determined by high time resolution electron paramagnetic resonance (EPR), performed at three different microwave frequencies. Structural information is extracted from light-induced quantum beats observed in the transverse magnetization of P700(+)A1(-) at early times after laser excitation. A computer analysis of the two-dimensional Q-band experiment provides the orientation of the various magnetic tensors of with respect to a magnetic reference frame. The orientation of the cofactors of the primary donor in the g-tensor system of is then evaluated by analyzing time-dependent X-band EPR spectra, extracted from a two-dimensional data set. Finally, the cofactor arrangement of P700(+)A1(-) in the photosynthetic membrane is deduced from angular-dependent W-band spectra, observed for a magnetically aligned sample. Thus, the orientation of the g-tensor of P700(+) with respect to a chlorophyll based reference system could be determined. The angle between the g1(z) axis and the chlorophyll plane normal is found to be 29 +/- 7 degrees, while the g1(y) axis lies in the chlorophyll plane. In addition, a complete structural model for the reduced quinone acceptor, A1(-), is evaluated. In this model, the quinone plane of is found to be inclined by 68 +/- 7 degrees relative to the membrane plane, while the P700(+)-A1(-) axis makes an angle of 35 +/- 6 degrees with the membrane normal. All of these values refer to the charge separated state, observed at low temperatures, where forward electron transfer to the iron-sulfur centers is partially blocked. Preliminary room temperature studies of P700(+)A1(-), employing X-band quantum beat oscillations, indicate a different orientation of A1(-) in its binding pocket. A comparison with crystallographic data provides information on the electron-transfer pathway in PSI. It appears that quantum beats represent excellent structural probes for the short-lived intermediates in the primary energy conversion steps of photosynthesis.

Cell Membrane↗
PubMed2001

A new spectrin, beta IV, has a major truncated isoform that associates with promyelocytic leukemia protein nuclear bodies and the nuclear matrix.

We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10-16 of beta-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length beta-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin betaIV, thus encodes a full-length minor isoform (SpbetaIVSigma1) and a truncated major isoform (SpbetaIVSigma5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpbetaIVSigma5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpbetaIVSigma5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin betaIV is the first beta-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds.

Amino Acid Sequence↗
PubMed2001

Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels.

Homologues of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca(2+) entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP(3) receptors (IP(3)Rs) for activation. Here we show that Trp4 and other Trp proteins also interact with IP(3)Rs. The IP(3)R-binding domain also interacts with calmodulin (CaM) in a Ca(2+)-dependent manner with affinities ranging from 10 nm for Trp2 to 290 nm for Trp6. In addition, other binding sites for CaM and IP(3)Rs are present in the alpha but not the beta isoform of Trp4. In the presence of Ca(2+), the Trp-IP(3)R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP(3)Rs inhibited the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 microm) but not a low (5 microm) concentration of the Trp-binding peptide of the IP(3)R. Our data support the view that both CaM and IP(3)Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP(3)Rs differ for each Trp.

Amino Acid Sequence↗
PubMed2001

Regional characteristics of sulfur and lead isotope ratios in the atmosphere at several Chinese urban sites.

Sulfur and lead isotope ratios in the atmosphere were measured at several selected sites (Harbin, Changchun, Dalian, Waliguan, Shanghai, Nanjing, Guiyang) in China and Tsukuba (Japan), to reveal regional sources characteristics over Eastern Asia. Average S isotope ratios for SO2 and sulfate in the atmosphere in China were close to those of the coals used in each region, indicating a considerable contribution of coal combustion to the sulfur compounds in the atmosphere. Most northern cities had around 5% sulfur isotope ratio, while Guiyang, a southwestern city in China, showed a considerably lower sulfur isotope ratio (about -3%) because of the unusually light sulfur isotope ratio of coals in this region. These were considerably different from the value (-1.4%) for Tsukuba (Japan). Lead isotope ratios also suggested that coal combustion considerably contributed to atmospheric lead in some cases in China. At the same time, influences by the emission of Chinese lead ores were also observed in northern cities. Seasonal variations of both sulfur and lead isotope ratios indicated the existence of a certain amount of industrial sources other than coal combustion. In addition, fractionation effect between SO2 and sulfate showed a seasonal tendency (high in winter (0-6%) and low in summer (-1-3%)), suggesting the oxidation pathway of SO2 changed seasonally.

Air Pollution↗