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Biomedical subjects

Y Yao

Publications and source records attributed to Y Yao.

At least 19 recordsLinked to original sources
PubMed1995

Quantal puffs of intracellular Ca2+ evoked by inositol trisphosphate in Xenopus oocytes.

1. Ca2+ liberation induced in Xenopus oocytes by a poorly metabolized derivative of inositol 1,4,5-trisphosphate (3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate; 3-F-InsP3) was visualized using a video-rate confocal microscope to image fluorescence signals reported by the indicator dye calcium green-1. 2. Low (10-30 nM) intracellular concentrations of 3-F-InsP3 evoked Ca2+ release as localized transient 'puffs'. Progressively higher concentrations (30-60 nM) gave rise to abortive Ca2+ waves triggered by puffs, and then (> 60 nM) to a sustained elevation of Ca2+ followed by the appearance of propagating Ca2+ waves. At concentrations up to that giving waves, the frequency of puffs increased as about the third power of [InsP3], whereas their amplitudes increased only slightly. 3. The rise of cytosolic Ca2+ during a puff began abruptly, and peaked within about 50 ms. The peak free Ca2+ level was about 180 nM, and the total amount of Ca2+ liberated was several attomoles (10(-18) mol), too much to be accounted for by opening of a single InsP3-gated channel. The subsequent decline of Ca2+ occurred over a few hundred milliseconds, determined largely by diffusion of Ca2+ away from the release site, rather than by resequestration. Lateral spread of Ca2+ was restricted to a few micrometres, consistent with an effective diffusion coefficient for Ca2+ ions of about 27 microns2 s-1. 4. The peak amplitudes of puffs recorded at a given site were distributed in a roughly Gaussian manner, and a small proportion of sites consistently gave puffs much larger than the main population. Intervals between successive puffs at a single site were exponentially distributed, except for a progressive fall-off in puffs seen at intervals shorter than about 10 s. Thus, triggering of puffs appeared to be stochastically determined after recovery from a refractory period. 5. There was little correlation between the occurrence of puffs at sites more than a few micrometres apart, indicating that puff sites can function autonomously, but closely (ca 2 microns) adjacent sites showed highly correlated behaviour. 6. Puffs arose from sites-present at a density of about 1 per 30 microns2 in the animal hemisphere, located within a narrow band about 5-7 microns below the plasma membrane. 7. We conclude that Ca2+ puffs represent a 'quantal' unit of InsP3-evoked Ca2+ liberation, which may arise because local regenerative feedback by cytosolic Ca2+ ions causes the concerted opening of several closely clustered InsP3 receptor channels.

Animals↗
PubMed1995

Calcium puffs in Xenopus oocytes.

The second messenger inositol 1,4,5-trisphosphate (InsP3) functions in large part by liberating calcium ions from intracellular stores. This release process is highly non-linear and shows a regenerative characteristic that allows production of all-or-none calcium spikes which propagate as waves. However, at low concentrations of InsP3 an additional mode of calcium liberation is seen in Xenopus oocytes, transient 'puffs' of cytosolic calcium that last for a few hundred milliseconds and are restricted to within a few micrometres. Puffs are generally of similar size and the amount of calcium released (about 3 x 10(-18) mol) suggests that they arise through the concerted opening of several InsP3-gated calcium release channels. Puff sites are present at a density of about one per 30 microns 2 in the animal hemisphere of the oocyte. Each site functions autonomously, producing puffs at largely random intervals. We conclude that calcium puffs represent 'quantal' units of InsP3-evoked calcium liberation, which may result from local regenerative feedback by cytosolic calcium ions at functionally discrete release sites.

Animals↗
PubMed1995

Acute administration of alpha-methyl-para-tyrosine alters levels of norepinephrine transporter mRNA in the rat brainstem.

This study investigated whether rat norepinephrine transporter (NET) mRNA levels would be altered by alpha-methyl-p-tyrosine (alpha-MPT), a tyrosine hydroxylase inhibitor. While NE levels decreased at 1 and 3 days but recovered at 7 days after alpha-MPT, NET mRNA levels decreased at 3 and 7 days but not at 1 day after alpha-MPT. The results indicate that acute treatment with alpha-MPT led to a delayed time response in its effects on NET mRNA and NE levels in the rat brain.

Actins↗
PubMed1995

Dioxin activates HIV-1 gene expression by an oxidative stress pathway requiring a functional cytochrome P450 CYP1A1 enzyme.

We have studied the effect of several environmental chemicals on the transient expression of a chloramphenicol acetyltransferase (cat) reporter gene linked to the promoter sequences in the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1). Aflatoxin B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and benzo[a]pyrene cause a significant increases in CAT expression in mouse hepatoma Hepa-1 cells. The induction of CAT after TCDD treatment is abolished by administration of N-acetyl-L-cysteine or 2-mercaptoethanol and does not take place in a mutant cell line that lacks CYP1A1 enzymatic activity. Linker-scanning mutational analysis of transcription factor binding sites in the promoter revealed that both the NF kappa B and an adjacent aromatic hydrocarbon response element (AhRE) are required for TCDD-dependent CAT expression. In addition, mutation of the NFAT/AP-1 binding sites in the negative regulatory region of the promoter increases the magnitude of the TCDD effect. We conclude that induction of a functional CYP1A1 monooxygenase by TCDD stimulates a pathway that generates thiol-sensitive reactive oxygen intermediates which, in turn, are responsible for the TCDD-dependent activation of genes linked to the LTR. These data might provide an explanation for findings that TCDD increases infectious HIV-1 titers in experimental systems and for epidemiologic reports suggesting that exposure to aromatic hydrocarbons, such as found in cigarette smoke, is associated with an acceleration in AIDS progression.

Amino Acid Sequence↗
PubMed1995

Therapeutic effects of the blood-activating and stasis-reducing method in 80 cases of coronary heart disease.

The pathogenesis and treatment of coronary heart disease (CHD) may vary, whereas the author believes that the disease is due to blood stasis on a fundamental basis of deficiency, and the principle of treatment is activation of blood. Since stasis may lead to deficiency and vice versa, CHD cannot be cured without removal of stasis, and stasis cannot be completely removed without proper correction of deficiency. The basic method for the treatment of CHD is therefore activation of blood for reduction of stasis. At the same time, according to the conditions of individual patients, the methods of replenishing Qi, warming Yang, nourishing Yin, reducing phlegm and subduing Yang should be applied simultaneously to resolve the problem of deficiency. The author treated 80 cases of CHD by the above rationale and found the results satisfactory, i.e., 28 cases (35%) showed marked symptomatic relief, 46 (57.5%) showed symptomatic improvement, while 6 cases (7.5%) failed. The total effective rate was 92.5%. ECG examination indicated 9 cases markedly effective, 27 improved, while 44 cases failed, for a total effective rate of 45%. Blood pressure and blood lipids also showed significant drops (P < 0.001).

Adult↗
PubMed1994

A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments.

Generation of soluble T-cell receptor (TCR) molecules by a variety of genetic engineering methods has been hampered by inefficient pairing of alpha and beta subunits in the absence of their respective transmembrane regions and associated CD3 components. To overcome this obstacle, we have added 30-amino acid-long segments to the carboxyl termini of alpha and beta extracellular domains via a cleavable flexible linker. These peptide segments (BASE-p1 for alpha and ACID-p1 for beta) have been previously shown to selectively associate to form a stable heterodimeric coiled coil termed a leucine zipper. Homodimeric structures are not permitted due to electrostatic repulsion among amino acid side chains. Expression of a representative TCR-leucine zipper fusion protein in a baculovirus expression system results in production of alpha beta TCR heterodimer at 0.6-1.4 mg/liter. This yield is 5- to 10-fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper sequence. The structure of the TCR component of the fusion heterodimer was judged to be native when probed with a panel of 17 mAbs specific for alpha and beta constant and variable domains. A mAb specific for the isolated BASE-p1/ACID-p1 coiled coil was also generated and shown to react with the TCR fusion protein. The above technology should be broadly useful in the efficient production and purification of TCRs as well as other heterodimeric proteins.

Amino Acid Sequence↗
PubMed1994

Resolution of structural changes associated with calcium activation of calmodulin using frequency domain fluorescence spectroscopy.

Structural changes associated with the calcium-dependent activation of wheat germ calmodulin (CaM) were assessed through measurements of steady-state and time-resolved changes in the fluorescence associated with (1) the unique tyrosine (Tyr139) located in calcium binding loop IV or (2) N-(1-pyrenyl)-maleimide (PM) or 4-(iodoacetamido)salicylic acid (IASA) covalently attached to Cys27 present in calcium binding loop I. These fluorophores permit the measurement of calcium-dependent changes in (i) the solvent accessibility and rotational dynamics associated with calcium binding loops I and IV and (ii) the hydrodynamic properties of the entire protein. Specific nitration of the unique tyrosine (Tyr139) in calcium binding loop IV permits the use of fluorescence resonance energy transfer to measure both the average spatial separation and distance heterogeneity between Cys27 and Tyr139, providing a direct measurement of the conformational flexibility of the central helix. Upon calcium binding, (i) the solvent accessibility and rotational dynamics of both PM and IASA (covalently bound to Cys27) and Tyr139 increase, (ii) overall protein rotational motion decreases, (iii) the average separation between the chromophores at Cys27 and nitrotyrosine 139 decreases, and (iv) the conformational flexibility associated with the central helix decreases. Therefore, upon calcium binding, the central helix becomes more extended and rigid, while the globular domains adopt a more open tertiary conformation that brings Cys27 and Tyr139 into closer proximity. This calcium-dependent structural change functions to expose the hydrophobic binding sites located within the globular domains, and to enhance the probability of binding target sequences through a reduction in conformational heterogeneity.

Calcium↗
PubMed1994

Ca2+ influx modulation of temporal and spatial patterns of inositol trisphosphate-mediated Ca2+ liberation in Xenopus oocytes.

Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger by liberating intracellular Ca2+ and by promoting influx of extracellular Ca2+. We examined the effects of Ca2+ influx on the temporal and spatial patterns of intracellular Ca2+ liberation in Xenopus oocytes by fluorescence imaging of cytosolic free Ca2+ together with voltage clamp recording of Ca(2+)-activated Cl- currents. Oocytes were injected with a poorly metabolized InsP3 analogue (3-F-InsP3; see Introduction) to induce sustained activation of InsP3 signalling, and Ca2+ influx was controlled by applying voltage steps to change the driving force for Ca2+ entry. Positive- and negative-going potential steps (corresponding, respectively, to decreases and increases in Ca2+ influx) evoked damped oscillatory Cl- currents, accompanied by cyclical changes in cytosolic free Ca2+. The source of this Ca2+ was intracellular, since oscillations persisted when Ca2+ entry was suppressed by removing extracellular Ca2+ or by polarization close to the Ca2+ equilibrium potential. Fluorescence recordings from localized (ca 5 microns) spots on the oocyte showed repetitive Ca2+ spikes. Their frequency increased at more negative potentials, but they became smaller and superimposed on a sustained 'pedestal' of Ca2+. Spike periods ranged from about 50 s at +20 mV to 4s at potentials between -60 and -120 mV. Ca2+ spike frequency decreased after removing extracellular Ca2+, but the spike amplitude was not reduced and low frequency spikes continued for at least 30 min in the absence of extracellular Ca2+. Membrane current oscillations decayed in amplitude following voltage steps, while locally recorded Ca2+ spikes did not. This probably arose because Ca2+ release was initially synchronous across the cell, leading to large Ca(2+)-activated Cl- currents, but the currents then diminished as different areas of the cell began to release Ca2+ asynchronously. Fluorescence imaging revealed that Ca2+ liberation in 3-F-InsP3-loaded oocytes occurred as transient localized puffs and as propagating waves. Polarization to more negative potentials increased the frequency of puffs and the number of sites at which they were seen, and enhanced their ability to initiate waves. The frequency and velocity of Ca2+ waves increased at more negative potentials. When the potential was returned to more positive levels, repetitive Ca2+ spikes at first occurred synchronously across the recording area, but this synchronization was gradually lost and Ca2+ waves began at several foci. We conclude that influx of extracellular Ca2+ regulates the temporal and spatial patterns of Ca2+ liberation from InsP3-sensitive intracellular stores, probably as a result of dual excitatory and inhibitory actions of cytosolic Ca2+ on the InsP3 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Decline of serum cobalamin levels with increasing age among geriatric outpatients.

The level of serum cobalamin declines with increasing age. Among 100 consecutive geriatric outpatients who were seen in office-based settings for various acute and chronic medical illnesses but not for cobalamin deficiency-related diseases, 11% had serum cobalamin levels from 148 to 295 pmol/L. The average annual serum cobalamin level decline was 18 pmol/L for patients who had higher initial serum cobalamin levels (actual range, from 224 to 292 pmol/L). The average annual serum cobalamin decline was 28 pmol/L, which was much higher, for patients who had lower initial serum cobalamin levels (actual range, from 157 to 221 pmol/L). Patients with initial serum cobalamin levels of 148 pmol/L or below received treatment immediately, and their declines could not be studied.

Aged↗
PubMed1994

Coexistence of fast-muscle-type and slow-muscle-type troponin T isoforms in single chimeric muscle fibers induced by muscle transplantation.

Regenerated muscle fibers which appeared after transplantation of chicken slow muscle (anterior latissimus dorsi) into breast fast muscle (pectoralis major) of the same animal were studied by two-dimensional SDS-polyacrylamide gel electrophoresis, immunoblotting, and immunostaining with antisera against fast-muscle-type troponin T and slow-muscle-type troponin T. In the transplanted muscle, degeneration of muscle fibers was followed by regeneration of slow muscle, which was revealed by detecting slow-muscle-type troponin T with the antiserum. Furthermore, coexistence of fast-muscle-type and slow-muscle-type troponin T isoforms in single chimeric muscle fibers composed of partly fast and partly slow fibers was observed in the regenerated muscle. We suggested that the chimeric fibers were originated from the fusion of fast and slow myoblasts during regeneration after muscle transplantation and that two nuclei differently determined in troponin T expression were working independently in a single cell.

Animals↗
PubMed1994

Persistent expression of tissue-specific troponin T isoforms in transplanted chicken skeletal muscle.

This study attempted to investigate the expression of skeletal muscle troponin T isoforms in chicken reared for six months after muscle transplantations of breast muscle into leg muscle, leg muscle into breast muscle, and slow muscle into breast and leg muscles of the same animal. The regenerated muscle after transplantation was studied by histological observation, two-dimensional SDS-polyacrylamide gel electrophoresis, and immunoblotting with anti-troponin T antibodies. Persistent expression of troponin T isoforms specific to donor tissue was observed in the regenerated muscle, and compared with their expression in the normal developing muscles. During the regeneration, the cells grew up and expressed troponin T isoforms in a manner similar to that in normal developing muscles, and on around the 178th day after the transplantation, the regenerated muscle expressed the adult type troponin T isoforms. Based on the troponin T isoforms expressed in the transplants, we consider that one type of skeletal muscle has some inherent potential to grow in and coexist with other types for a long term.

Animals↗
PubMed1994

Relation between intracellular Ca2+ signals and Ca(2+)-activated Cl- current in Xenopus oocytes.

Activation of inositol 1,4,5-trisphosphate (InsP3) signalling in Xenopus oocytes causes intracellular Ca2+ mobilization and thereby activates a Ca(2+)-dependent Cl- membrane conductance. Measurements of cytosolic Ca2+ levels using fluorescent indicators, however, revealed little correspondence with Cl- currents. Intracellular photorelease of InsP3 from a caged precursor evoked transient currents that peaked while the Ca(2+)-fluorescence signal was rising, and subsequently declined within a few seconds, even though the Ca2+ signal remained elevated much longer. Also, Cl- currents evoked by agonist activation showed transient spikes while a wave of Ca2+ liberation swept across the cell, but then decreased when the Ca2+ signal attained a maximal level. Thus, the Cl- current corresponded better to the rate of rise of intracellular free Ca2+, rather than to its steady state level. Experiments using paired flashes to photolyse caged InsP3 and caged Ca2+ indicated that this relationship did not arise through desensitization or inactivation of the Cl- conductance. Furthermore, fluorescence measurements made at different depths into the cell using a confocal microscope revealed no evidence that a rapid decline of local Ca2+ levels near the plasma membrane was responsible for the decay of Ca(2+)-activated Cl- current. Instead, Cl- channels may show an adaptive or incremental response to Ca2+, which is likely to be important for the encoding and transmission of information by Ca2+ spikes.

Animals↗
PubMed1994

The individual microbiologic effect of three antimycobacterial agents, clofazimine, ethambutol, and rifampin, on Mycobacterium avium complex bacteremia in patients with AIDS.

The individual antibacterial activities of clofazimine, ethambutol, and rifampin in the treatment of Mycobacterium avium complex bacteremia in patients with AIDS were determined. Sixty human immunodeficiency virus 1-infected patients who had at least one blood culture positive for M. avium complex were randomized to receive either clofazimine (200 mg), ethambutol (15 mg/kg), or rifampin (600 mg) once daily for 4 weeks. Only ethambutol resulted in a statistically significant reduction in the level of mycobacteremia. The median change in individual baseline colony counts was -0.60 log10 cfu/mL after 4 weeks of ethambutol (P = .046). In contrast, median changes in individual baseline colony counts were -0.2 log10 cfu/mL and +0.2 log10 cfu/mL for clofazimine and rifampin, respectively (both, P > .4). Ethambutol had greater antibacterial activity, as determined by changes in the level of mycobacteremia, than either rifampin or clofazimine, supporting its continued use in combination with other agents in the treatment of M. avium infection.

AIDS-Related Opportunistic Infections↗
PubMed1994

Rapid detection of phenylketonuria mutations by non-radioactive single-strand conformation polymorphism analysis.

A non-radioactive single-strand conformation polymorphism (SSCP) method was used to detect various phenylketonuria (PKU) mutations in Japanese and Chinese patients. Arginine413-to-proline (R413P) mutation in exon 12 of the phenylalanine hydroxylase gene was identified in a Japanese patient by this method. The segregation of the R413P mutation in the proband's family was clearly demonstrated and the carrier status of each family member was determined. Analysis of DNA fragments containing exon 7 originated from Chinese patients revealed two mutations, arginine243-to-glutamine (R243Q) and arginine261-to-glutamine (R261Q), and a polymorphism, valine245-to-valine (V245V). Although R261Q has been identified previously among Caucasian subjects, this report is the first to describe this mutation among Orientals. Since the non-radioactive SSCP method employs pre-cast acrylamide gels and pre-made gel buffer strips combined with semi-automated temperature-controlled electrophoresis, it can be performed without much expertise in molecular biological techniques. The ability of this method to detect various mutations as demonstrated in this study and its ease of use make it feasible to detect PKU mutations in a routine DNA diagnostic laboratory.

Asian People↗
PubMed1994

[Effects of interferon on infant with CMV hepatitis].

Fifty-seven patients with CMV hepatitis, who were within 3 months of age, were treated with interferon from August 1992 to April 1993. They were randomized to be assigned to two groups. There were 30 patients in the therapeutic group, who were given daily interferon 1,000,000 u intramuscularly for 10 days and 27 patients in the control group, who were put on daily vitamin C and inosine therapy intravenously for 10 days. The results showed that there was a significant difference in the aspects of jaundice disappearance, liver and serum SGPT recovery time between the two groups. In the therapeutic group, the average alleviating time of jaundice, hepatomegaly and serum SGPT level after treatment with interferon were 15.26, 29.89 and 14.26 days ahead of those in the control group, respectively. The cure rate at one month and two months after treatment was 43.0% and 90.0% in the therapeutic group, respectively; 18.5% and 48.0% in the control group, respectively.

Cytomegalovirus Infections↗
PubMed1994

[The role tumor necrosis factor in multiple organ dysfunction caused by bowel ischemia and reperfusion].

To explore the role of tumor necrosis factor (TNF) in the pathogenesis of multiple organ dysfunction following bowel ischemia and reperfusion, 98 rats were subjected to occlusion of the superior mesenteric artery for 45 minutes. It was found that the plasma TNF level increased rapidly after release of the clamp, peaking to 27.59 +/- 11.13 ng/ml 2 hours after reperfusion. Its changes in quantity was directly related to endotoxin in the portal circulation. Furthermore, the results showed that pretreatment with monoclonal antibody to TNF-alpha could significantly lowered the plasma TNF content and notably improved the functions of various organs. This study demonstrated that release of TNF might result in systemic hypotension and remarkable damage to liver, kidneys and lungs, which contributed to the development of sepsis and multiple system organ failure following severe ischemia-reperfusion injury of the intestine.

Animals↗