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Biomedical subjects

J Tao

Publications and source records attributed to J Tao.

At least 91 records · Page 5Linked to original sources
PubMed1994

Human alpha atrial natriuretic peptide fermentation by using a genetically engineered yeast strain.

The genotype of the recombinant yeast strain Y33::YFD71-3 used in this study is alpha, his, leu, ade and suc. Preliminary batch cultures in shaking flasks showed the expression level of atrial natriuretic peptide (ANP) was 1-2 mg/L. Shaking flask cultures were carried out in YG medium which contained glucose, yeast nitrogen base (YNB), and different amounts of adenine, histidine and leucine. When the growth was limited by adenine, protein secreted from Y33::YFD71-3 cells was increased obviously. Adenine became the limiting substrate when the YG medium was supplemented with 5 g/L of casamino acid (CAA) and the level of ANP expression was influenced by the concentrations of adenine, YNB and leucine in the medium. In fed batch cultures carried out in a RIBE-5 fermenter, the cultures were fed with glucose and a mixture of YNB, CAA, adenine, histidine and leucine to improve cell growth and ANP expression, and the maximum ANP concentration in the culture reached 24.8 mg/L.

Adenine↗
PubMed1993

Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins.

The transcriptional activating (Tat) proteins from human immunodeficiency virus and simian immunodeficiency virus are sequence-specific RNA-binding proteins. In human immunodeficiency virus Tat, a single arginine residue, flanked on each side by three to four basic amino acids, mediates specific binding to a bulge region in trans-acting responsive element (TAR) RNA. We have systematically mutated the flanking charged residues and found that, in addition to the position of the sequence-specific arginine, the particular arrangement of nonspecific electrostatic interactions is an important determinant of RNA-binding specificity and transactivation activity. These additional electrostatic contacts may help stabilize the structure of TAR RNA when bound to arginine. One critical electrostatic interaction, located two residues N-terminal to the arginine, is absent in the simian immunodeficiency virus Tat protein and accounts for the difference in promoter specificities of the human and simian immunodeficiency viral proteins.

Amino Acid Sequence↗
PubMed1993

Evaluation of mouse sperm acrosomal status and viability by flow cytometry.

A procedure was developed to evaluate mouse sperm acrosomal status and viability simultaneously utilizing flow cytometry. Four fluorescein isothiocyanate (FITC)-conjugated lectins, peanut agglutinin (PNA), concanavalin agglutinin (ConA), Pisum sativum agglutinin (PSA), and soybean agglutinin (SBA), were investigated, with PNA providing the greatest sensitivity and specificity in distinguishing acrosome-present and acrosome-absent mouse spermatozoa. To expose lectin binding sites, digitonin (20 microM at room temperature for 10 min) was used to permeabilize sperm plasma membranes. Sperm cell viability was determined by Hoechst 33258-(H258) exclusion. To prevent permeabilized cells from staining with H258, salmon sperm DNA (SS-DNA) was applied to bind excess dye in the solutions after supravital staining. Calcium ionophore (A23187; 5 or 20 microM) was used to induce acrosome reactions. The results of flow cytometric analyses were compared with epifluorescence microscopic observation and were highly correlated (r = 0.999; P < 0.001). The method developed provides an objective and efficient procedure to estimate simultaneously both acrosomal status and viability of mouse spermatozoa.

Acrosome↗
PubMed1993

Mutants of Ustilago maydis defective in production of one of two polypeptides of KP6 toxin from the preprotoxin.

Double-stranded RNA viruses of Ustilago maydis encode secreted killer toxins to which other cells of the same species and closely related species are sensitive. KP6 toxin consists of two polypeptides, alpha and beta, produced from a single precursor preprotoxin. In this work, we cloned complementary DNA for the toxin-encoding segment of two of the KP6 nonkiller mutants NK3 and NK13 that secrete the beta and alpha polypeptides, respectively. Both sequence analysis of the cDNA clones and in vitro translation of the toxin-encoding double-stranded RNAs showed that both mutants can produce full-length preprotoxins. Cys51 in alpha is converted to Arg in NK3 and Thr25 and Lys42 in beta are changed to Pro and Arg, respectively, in NK13. Although alpha and beta are encoded in a single prepropolypeptide, only the beta polypeptide is secreted by NK3 and only the alpha polypeptide is secreted by NK13. This differential expression of peptides from one precursor is a unique phenomenon. Neither of the nonsecreted polypeptides accumulated in the cytosol. The possible effects of these mutations on preprotoxin folding and their consequences for toxin secretion are discussed.

Amino Acid Sequence↗
PubMed1993

Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry.

Acrosomal status and viability were evaluated simultaneously on human spermatozoa using flow cytometry. Samples were divided into three aliquots and randomly assigned to one of three treatments: (i) cryopreservation; (ii) 10 microM calcium ionophore [A23187 in dimethylsulphoxide (DMSO)] or (iii) DMSO alone (control). Acrosomal status was evaluated using monoclonal antibodies recognizing MH61 and CD46, respectively. Fluorescein-conjugated goat anti-mouse immunoglobulin (IgG) was used as a second antibody. Sperm viability was assessed using Hoechst 33258 (H258) exclusion. The following factors were analysed: (i) the specificity of the monoclonal antibodies for the human acrosome; (ii) the relative effectiveness of flow cytometry and direct fluorescent microscopy scoring and (iii) the acrosomal status and viability of the control, ionophore-treated, and cryopreserved spermatozoa. Across all treatments, the MH61 and CD46 monoclonal antibodies resulted in acrosomal status values (acrosome-reacted/viable spermatozoa) which were not significantly different (P > 0.05): control, 1.0 +/- 0.3% and 1.5 +/- 0.6% (mean +/- SEM); A23187, 42.8 +/- 3.5% and 38.1 +/- 3.5%; cryopreserved, 8.2 +/- 2.0% and 9.9 +/- 1.3%; respectively. However, acrosomal status among treatments differed significantly (P < 0.01). Flow cytometric and direct fluorescent microscopy assessments were significantly correlated (r2 = 0.96, P < 0.01). These results indicate that flow cytometry, using an acrosome-specific monoclonal antibody and a supravital dye, provides an objective and efficient method to evaluate human sperm acrosomal and viability status simultaneously.

Acrosome↗
PubMed1993

Possibility of signal transduction through microfilaments below the membrane following ligand-receptor interaction.

In this paper, we describe the changes of microfilament assembly and 3H-TdR incorporation in mouse ascites liver cancer cells under the action of concanavalin A (ConA) and laminin (LN). We have also studied the variation of 3H-TdR incorporation induced by destroying microfilaments with cytochalasin B (CB) following ConA and LN binding with their membrane receptors. It was found that ConA and LN interactions with their membrane receptors could induce the assembly of microfilaments below the membrane and promote DNA synthesis in these cells, but this effect was inhibited when microfilaments were destroyed by CB treatment. These results suggest that microfilaments might play a role in transferring signals from the membrane to the nucleus.

Actin Cytoskeleton↗
PubMed1993

The effect of laminin on molecular motion in the cell membrane and on cell motility.

We have studied the variation of lateral diffusion of proteins in the cell membrane, of membrane lipid fluidity and of the electrophoretic motility (EPM) of macrophages after treatment with extrinsic laminin. The results showed that the lateral diffusion coefficient D value of membrane proteins, the fluidity of membrane lipids and the EPM of macrophages were decreased after laminin had bound to its membrane receptor on the macrophages. These results are important for developing an understanding of the early reaction of plasma membranes and cells in the presence of laminin.

Animals↗
PubMed1992

Actions of thapsigargin on the Ca(2+)-handling systems of the human platelet. Incomplete inhibition of the dense tubular Ca2+ uptake, partial inhibition of the Ca2+ extrusion pump, increase in plasma membrane Ca2+ permeability, and consequent elevation of resting cytoplasmic Ca2+.

Thapsigargin (Tg) effects on Ca2+ handling in the intact human platelet were studied using Quin2 and chlorotetracycline to measure free cytoplasmic and dense tubular (DT) Ca2+ concentrations ([Ca2+]cyt and [Ca2+]dt, respectively). Tg inhibits Ca2+ uptake by the DT Ca(2+)-ATPase pumps, but incompletely, lowering the Vm to 32% of control (IC50,Tg = 0.18 +/- 0.10 microM). The kinetics of loss of DT Ca2+, transient increases in [Ca2+]cyt, and lowered steady-state [Ca2+]dt after Tg addition are all explained by pump inhibition, with no effect on the rate constant of Ca2+ leakage across the DT membrane (kleak,DT = 1.14 min-1). Tg lowers by 30% the Vm of the Ca2+ extrusion pump located in the plasma membrane (PM), as shown by a Quin2-based method measuring active Ca2+ extrusion (Johansson, J. S., and Haynes, D. H. (1988) J. Membr. Biol. 104, 147-163). This effect (IC50,Tg = 0.45 +/- 0.06 microM), together with a 24 +/- 16% increase in kleak,PM,Ca (to 3 x 10(-4) min-1), accounts for a Tg-dependent sustained elevation [Ca2+]cyt (to 708 +/- 78 nM) which is independent of DT Ca2+ status or history. Thrombin and Tg release 30 and 70% (respectively) of the DT Ca2+ available at any instant, independent of order of challenge, consistent with a single class of DT with respect to these agents.

Biological Transport↗
PubMed1992

Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C.

The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.

Blood Platelets↗
PubMed1992

Specific binding of arginine to TAR RNA.

A single arginine residue within the basic region of the human immunodeficiency virus Tat protein mediates specific binding of Tat peptides to a three-nucleotide bulge in TAR RNA. It has been proposed that arginine recognizes TAR by forming a network of hydrogen bonds with two structurally distinct phosphates, an interaction termed the "arginine fork." Here it is shown that L-arginine blocks the Tat peptide/TAR interaction, whereas L-lysine and analogs of arginine that remove specific hydrogen bond donors do not. Experiments using an L-arginine affinity column demonstrate that arginine and the Tat peptides bind to the same site in TAR. Modification of two phosphates located at the junction of the double-stranded stem and bulge and modification of two adenine N7 groups in base-paired regions of TAR interfere with specific arginine binding. The results emphasize the importance of RNA structure in RNA-protein recognition and provide methods to identify arginine-binding sites in RNAs.

Adenine↗
PubMed1992

Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP.

Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).

Biological Transport↗
PubMed1992

Immunity and resistance to the KP6 toxin of Ustilago maydis.

The KP6 toxin of Ustilago maydis, encoded by segmented double-stranded (ds) RNA viruses, is lethal to sensitive strains of the same species and related species. The toxin consists of two polypeptides, alpha and beta, synthesized as a single preprotoxin, which are not covalently linked. Neither polypeptide alone is toxic, but killer activity can be restored by in vitro and in vivo complementation. Killer-secreting strains are resistant to the toxin they produce. Resistance is conferred by a single recessive nuclear gene. This study describes a search for cytoplasmic factors that may confer resistance, also referred to as immunity. The approaches used to detect cytoplasmic immunity included transmission of dsRNA and transmission of virus particles to sensitive cells by cytoduction, cytoplasmic mixing in diploids and infection with viruses. An alternative approach was also used to express cloned cDNAs of the KP6 toxin-encoding dsRNA and of the alpha and beta polypeptides. The results indicated that no immunity to KP6 can be detected. While KP6, alpha and beta polypeptides were expressed by resistant cells, neither KP6 nor beta were expressed in sensitive strains. The alpha polypeptide was expressed in sensitive cells, but it did not confer immunity. These results suggest that neither the preprotoxin nor the alpha or beta polypeptides confer immunity and thus beta may be the toxic component of the binary toxin.

Blotting, Northern↗
PubMed1990

Experimental and clinical studies on inhibitory effect of ganoderma lucidum on platelet aggregation.

In this study we observed the inhibitory effect of Chinese herbal medicine Ganoderma lucidum (GL) on platelet aggregation in 15 healthy volunteers and 33 patients with atherosclerotic diseases. The results showed that the first and the second phase of aggregation of platelets of the healthy volunteers were obviously inhibited (P less than 0.01) when watery soluble extract of GL of different concentrations was added to the platelets in vitro, i. e., the reaction speed of platelet aggregation was slowed down. The inhibitory effect was related to dosage. Platelet aggregation induced by ADP in final concentration of 2 mumol/L and 3 mumol/L was obviously inhibited, after the patients had taken GL 1 g 3 times a day for 2 weeks, the maximum platelet aggregation inhibition rates were then 31.49% (P less than 0.01) and 17.7% (P less than 0.01) respectively. Length and weights (wet and dry) of the extracorporeal thrombi were reduced from 30.05 +/- 4.38 mm, 103.9 +/- 9.33 mg and 44.89 +/- 4.79 mg to 20.4 +/- 2.33 mm (P less than 0.05), 85.27 +/- 8.77 mg (P less than 0.01) and 35.1 +/- 4.5 mg (P less than 0.01) respectively after oral administration of GL. The results of our experiments suggested that the Chinese herbal medicine GL may be an effective inhibitory agent of platelet aggregation. However, its mechanism and active principles remain to be further investigated.

Aged↗
PubMed1990

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.

Amino Acid Sequence↗
PubMed1986

Projections from the hypothalamus and its adjacent areas to the posterior pituitary in the rat.

Cholera toxin conjugated horseradish peroxidase was injected into the posterior pituitary and its afferents traced in 21 albino rats. The neuronal processes as well as the perikarya were elaborately displayed. The principal and retrochiasmatic supraoptic nuclei and the magnocellular paraventricular subnuclei were densely labelled. The accessory cell groups or nuclei labelled included: the medial preoptic and anterior hypothalamic areas, the anterior and posterior fornical nuclei, the lateral hypothalamic area, the nucleus circularis and nucleus of the forebrain bundle and hitherto unknown or not fully appreciated retrochiasmatic area, the dorsal accessory groups in an area between the stria medullaris and fornix, on the one hand, and the stria terminalis and internal capsule, on the other, and a well developed subependymalperiventricular zone. The medial preoptic nucleus, subfornical organ and organ vasculosum laminae terminalis were also weakly stained. Dendrites of the magnocellular paraventricular nucleus have been said by some to be largely confined to the subnuclei in which they lie. Immunohistochemical studies have proved that they extended beyond their nuclear confinement. The present study has found much wider extension of their dendritic fields. In fact, dendrites of the magnocellular neurosecretory cells in general were long and had a certain degree of directional bias. Several sites projecting to the posterior pituitary were closely related to the cerebrospinal fluid. Namely, the subependymal neuronal plexuses along the third ventricle and beneath the interventricular foramen, and the subpial dendritic plexuses of the supraoptic and retrochiasmatic supraoptic nuclei. Neurons were seen to squeeze in-between the ependymal cells, bringing themselves very close to the cerebrospinal fluid. No direct cerebrospinal fluid-contacting elements, either cell bodies or processes, however, could be ascertained. It is proposed that these plexuses may monitor changes in the cerebrospinal fluid. Besides the principal neurohypophysial tract the posterior pituitary was found in the present study to receive its afferents via two accessory fasciculi, one coursing in the medial forebrain bundle and the other running along the lateral wall of the infundibular recess subependymally.

Afferent Pathways↗