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Biomedical subjects

J Tao

Publications and source records attributed to J Tao.

At least 73 records · Page 4Linked to original sources
PubMed1997

Dissection of the proposed base triple in human immunodeficiency virus TAR RNA indicates the importance of the Hoogsteen interaction.

A single arginine residue within the RNA-binding domain of the human immunodeficiency virus Tat protein makes a critical sequence-specific contact to TAR RNA. Arginine as the free amino acid also binds specifically to TAR and induces a change in RNA conformation similar to that induced by Tat peptides. NMR and biochemical studies have suggested that the arginine-binding site is stabilized by a base triple interaction between a bulged U and an A x U base pair in the adjacent stem. In this study, we have used chemical modification and mutagenesis experiments to examine the relative contributions of the Watson-Crick and Hoogsteen base-pairing partners of the proposed U-A x U base triple. We show that the Hoogsteen interaction is critical for arginine binding whereas the Watson-Crick interaction can be eliminated or replaced by other base-base interactions. The results are consistent with biochemical studies of the Tat-TAR interaction and support the base triple model for the structure of TAR.

Arginine↗
PubMed1997

Suppression of human ribosomal protein L23A expression during cell growth inhibition by interferon-beta.

Interferons inhibit cell growth in normal and tumor-derived cells. The molecular basis of interferons antiproliferative activity remains to be defined. Using subtraction hybridization, a human melanoma differentiation associated gene, mda-20, has been identified that is down-regulated by treatment with interferon. Sequence analysis indicates that mda-20 is human ribosomal protein L23a (rp L23a). The mRNA levels of rp L23a and growth are diminished in a variety of human tumor cell lines following treatment with human fibroblast interferon, interferon-beta (IFN-beta). Expression of rp L23a is also reduced in human melanoma cells treated with human leukocyte (IFN-alpha) and immune (IFN-gamma) interferons, but not by growth inhibition resulting from serum starvation. These findings suggest that growth suppression alone is not sufficient to reduce rp L23a expression. Instead, reduced rp L23a mRNA results from biochemical changes mediated by interferons. Ectopic expression of an antisense rp L23a sequence in human HeLa cervical carcinoma cells results in a reduction in colony formation indicating a direct antiproliferative effect by inhibiting rp L23a expression. The mechanism underlying inhibition in rp L23a expression in IFN-beta-treated cells may involve antisense rp L23a RNA. These results suggest that rp L23a may be one of the target molecules involved in mediating growth inhibition by interferon.

Adenocarcinoma↗
PubMed1997

Basic fibroblast growth factor, its high- and low-affinity receptors, and their relationship to form-deprivation myopia in the chick.

Form deprivation myopia in chickens is a widely accepted model to study visually-regulated postnatal ocular growth. Recently we showed that basic fibroblast growth factor-2 provides a "stop" signal for the growing eye. To understand further its action, we have localized basic fibroblast growth factor-2 and its low- and high-affinity receptors in the chicken eye, and determined the localization of basic fibroblast growth factor receptors in the inner plexiform layer with respect to that of neurotransmitter systems known to play a role in form-deprivation myopia. By immunocytochemistry and in situ hybridization, two complementary methods, we found that nearly all cells in the retina, and scleral chondrocytes, contain basic fibroblast growth factor-2 protein and messenger RNA as well as high-affinity basic fibroblast growth factor receptor protein and messenger RNA. Immunocytochemical localization of basic fibroblast growth factor-2 binding sites (a high resolution alternative to autoradiography), combined with N-glycanase and heparitinase treatment or heparin competition, revealed additional binding sites in specific synaptic layers of the inner plexiform layer and low-affinity binding sites in the choroid and optic fibre layer. Some binding sites in the synaptic layers were found to co-stratify with neurites of dopamine-, vasoactive intestinal polypeptide- or enkephalin-containing amacrine cells, suggesting that basic fibroblast growth factor-2 could modulate synaptic transmission to or from these cells. Form deprivation did not affect the levels of basic fibroblast growth factor receptor-1 messenger RNA in retina/retinal pigment epithelium/choroid (Northern blotting), but it abolished the decrease in amount of extractable basic fibroblast growth factor normally observed in the dark (Western blotting). The results are discussed with respect to previous findings on basic fibroblast growth factor-2 and basic fibroblast growth factor receptor-1 localization in the avian and other vertebrate eyes, and their relevance to form-deprivation myopia. The widespread distribution of basic fibroblast growth factor-2 and its receptor makes it impossible to predict which cells might mediate the action of basic fibroblast growth factor-2 in form-deprivation myopia. However, the alteration in amounts of extractable retinal basic fibroblast growth factor-2 in form-deprived, dark-adapted retinas, in which basic fibroblast growth factor-2 probably serves as a "stop" signal for ocular growth, is consistent with a role for basic fibroblast growth factor-2 in the regulation of ocular growth.

Animals↗
PubMed1997

Pharmacokinetics of insect repellent N,N-diethyl-m-toluamide in beagle dogs following intravenous and topical routes of administration.

The common topical insect repellent N,N-diethyl-m-toluamide (DEET) has caused serious adverse effects in the users of DEET products due to its high skin permeability. This study investigated the pharmacokinetics of DEET following i.v. and dermal routes of administration in beagle dogs. The pharmacokinetics of DEET was linear over the dose range of 2.5-6.0 mg/kg. Following the i.v. dosing, plasma DEET concentrations declined biexponentially with an elimination half-life of 2.56 h. Volume of distribution at steady-state, systematic clearance, and mean residence time were estimated as 6.21 L/kg, 2.66 L/h/kg, and 2.34 h, respectively, indicating that DEET underwent extensive extravascular distribution and rapid elimination. After the dermal application of a commercial lotion and a new DEET lotion at 15 mg of DEET/kg, plasma DEET concentrations peaked at 1-2 h postdose. The DEET transdermal bioavailability and mean absorption time were 18.3% and 2.05 h, respectively, for the commercial lotion and 14.0% and 2.66 h, respectively, for the new lotion. The difference in DEET transdermal absorption between the two lotions suggested that commercial DEET products could be optimized for reduced DEET absorption for safer use.

Administration, Cutaneous↗
PubMed1997

[Mutation and methylation of CDKN2 gene in human head and neck carcinomas].

OBJECTIVE: To study mutations of CDKN2 gene in human head and neck carcinomas (HNC) and to elucidate the role of this gene in tumorigenesis. METHODS: PCR-SSCP and sequencing techniques were used to detect mutations in fresh specimens from 36 HNC cases. Specimens from 20 cases were further analysed for methlyation status of the CpG islands in the first exon of the gene using restriction enzyme digestion plus Southern blotting analysis. RESULTS: CDKN2 gene mutations were detected in 4 HNC cases of which 2 were missense mutation and the other 2 were frameshift mutation. Methylation status analysis revealed that 8 out of 20 HNC cases had de novo methylation of CpG island within the first exon of CDKN2 gene. CONCLUSION: The results shown here suggest that in addition to gene mutation and deletion, de novo methylation of the CpG island may be a frequent event in human carcinogenesis.

Carcinoma, Squamous Cell↗
PubMed1996

Stimulation of stress-activated protein kinase and p38 HOG1 kinase in murine keratinocytes following photodynamic therapy with benzoporphyrin derivative.

The activation state of the members of the mitogen-activated protein kinase family following photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A was investigated using a naturally transformed murine keratinocyte cell line, Pam 212. PDT involves the use of photosensitizer molecules and a specific wavelength of visible light. The process of PDT generates singlet oxygen and other reactive oxygen intermediates (ROIs), and the cytotoxic effect of these ROIs is the basis for the use of PDT to treat cancer and psoriasis. PDT caused a strong dose- and time-dependent activation of both stress-activated protein kinase (SAPK) and p38 HOG1. The maximum activation of SAPK and p38 HOG1 occurred between 20 and 30 min following PDT treatment with 200 ng/ml benzoporphyrin derivative monoacid ring A and 2 J/cm2 of red light at 690 nm. In our system, PDT did not cause significant activation of extracellularly regulated kinase (ERK) 1 and ERK2. Under the same experimental conditions, ultraviolet light irradiation caused strong activation of SAPK and p38 HOG1 and minimum activation of ERK1 and ERK2 in Pam212 cells. A number of ROI scavengers were tested for their effect on PDT-induced SAPK and p38 HOG1 activation. Both L-histidine and N-acetyl-L-cysteine showed a significant inhibitory effect on PDT-induced SAPK and p38 HOG1 activation. This indicated that PDT-induced SAPK and p38 HOG1 activation may be partially mediated by ROI.

Animals↗
PubMed1996

Variability of the thrombin- and ADP-induced Ca2+ response among human platelets measured using fluo-3 and fluorescent videomicroscopy.

The intracellular free Ca2+ concentration ([Ca2+]cyt) of individual human platelets localized between siliconized glass cover slips was determined at rest and after stimulation with thrombin and ADP using the Ca2+ indicator fluo-3 (0.97 +/- 0.30 mmol/l cell volume) with fluorescence video microscopy. Resting [Ca2+]cyt in the presence of 2 mM external Ca2+ showed only small inter-platelet variability ([Ca2+]cyt = 86 +/- 30 (S.D.) nM). Resting [Ca2+]cyt of individual fluo-3-loaded platelets measured as a function of time had a S.D. of 10 nM or 12% (S.D./mean). Individual platelets showed no affinity for the siliconized support and their [Ca2+]cyt showed no tendency to oscillate in either the resting or in the activated state. When 0.2 U/ml thrombin or 20 microM ADP were added, all platelets showed a characteristic Ca2+ transient whereby [Ca2+]cyt increased to peak values within 8-12 sec and then declined. The Ca2+ transients measured with fluo-3 were in approximate synchrony but peak [Ca2+]cyt values showed large inter-platelet variability. The ensemble average peak [Ca2+]cyt for thrombin and ADP were 672 +/- 619 (S.D.) nM and 640 +/- 642 (S.D.) nM, respectively. Thus inter-platelet variations (S.D./mean) were 92% or 100% as large as the average measured values. Mathematically-constructed averages of the single platelet experiments agreed reasonably well with platelet-averaged values obtained in parallel experiments with stirred platelet suspensions in a plastic cuvette, measured with a conventional spectrofluorometer. Peak [Ca2+]cyt values reflecting dense tubular Ca2+ release alone (external Ca2+ removed) also showed large interplatelet variation (171 +/- 105 (S.D.) nM with thrombin and 183 +/- 134 (S.D.) nM with ADP). Dense tubular Ca2+ release induced by cyclopiazonic acid (a dense tubular Ca2+-ATPase inhibitor) gave peak [Ca2+]cyt of 289 +/- 170 nM. Thus the size of the dense tubular Ca2+ pool has an inter-platelet variation of 59% (S.D./mean). Variability of the dense tubular pool size accounts for some, but not all, of the large interplatelet variation in peak (Ca2+]cyt seen with thrombin and ADP activation.

Adenosine Diphosphate↗
PubMed1996

Arginine-binding RNAs resembling TAR identified by in vitro selection.

Specific binding of the human immunodeficiency virus Tat protein to its RNA site (TAR) is mediated largely by a single arginine residue located within a basic region of the protein. Many essential features of the interaction can be mimicked by the free amino acid arginine, and an NMR model has been proposed in which the arginine guanidinium group binds to a guanine base in the major groove and to two phosphates adjacent to a bulge, with the RNA structure stabilized by a base triple between a U in the bulge and an adjacent A:U base pair. To compare the TAR structure to other arginine-binding RNAs, we performed in vitro selection experiments and identified RNAs with arginine-binding affinities similar to TAR. About 40% of the selected RNAs contained the same motif found in TAR: two stems separated by a bulge of at least two nucleotides, a U at the 5' position of the bulge, and G:C and A:U base pairs above the bulge. In many cases, the upper stems contained only the G:C and A:U pairs, located next to small loops. Chemical modification experiments demonstrated that these "TAR-like" RNAs bound arginine in a manner similar to TAR, and in some cases identified nucleotides outside the binding site that contributed to binding. To explore how small loops might help stabilize the structures of adjacent arginine-binding sites, we measured arginine-binding affinities of TAR-like RNAs having all possible three-nucleotide loops. An RNA with a UAG loop bound with highest affinity, and chemical modification and RNase mapping experiments suggested that the RNA changes conformation upon arginine binding, converting a large unstructured loop into a bulge conformation related to that of TAR. The results suggest that the arginine-binding site in TAR is structurally versatile and demonstrate how binding can be modulated by the surrounding RNA context.

Arginine↗
PubMed1996

Osmotic properties of boar spermatozoa and their relevance to cryopreservation.

A series of six experiments was conducted to determine the fundamental cryobiological properties of boar spermatozoa to develop optimal approaches for cryopreserving this important cell type. In the first experiments, boar spermatozoa samples were diluted in various osmolalities of experimental solutions (185-900 mOsmol kg-1) to provide hypo-, iso-, and hyperosmotic conditions. Equilibrium cell volumes (Expts 1 and 2) were measured after exposure for 3 min and the change in cell volume was measured over time using an electronic particle counter (Expt 3). The isosmotic cell volume was found to be 26.3 +/- 0.39 microns 3 (mean +/- SEM; n = 5). Over this range of osmolalities, boar spermatozoa behaved as linear osmometers (a linear volume versus 1/osm plot, r2 = 0.99) with an osmotically inactive cell fraction of 67.4 +/- 4.5%. The rate of water permeability (Lp) was determined to be 1.03 +/- 0.05 microns min-1 atm-1, which was consistent within and among donors (P > 0.130). A second series of experiments was performed to determine the effect of temperature and osmolality on boar sperm motility (Expt 4), and the effect of osmolality on the integrity of the sperm plasma membrane and its temperature dependence. Plasma membrane integrity was measured before and after boar spermatozoa were returned to an isosmolality (Expt 6). Motility was not affected at 30 degrees C, relative to that at room temperature, but was significantly decreased (P < 0.05) at 8 degrees C and 0 degree C (yielding a relative reduction to 85% and 35% of original motility, respectively; n = 6). Sperm motility was not significantly decreased (P > 0.05) until the osmolality reached 210 mOsmol kg-1, at which time motility began to decrease from 95% to 10% of the original value at 90 mOsmol kg-1. The integrity of the plasma membrane of boar spermatozoa was found to be dependent on temperature, donor and osmolality, decreasing significantly (P < 0.05) below room temperature, and below 185 mOsmol kg-1 (P < 0.05). There was no significant difference (P > 0.10) in the integrity of the plasma membrane of the samples before and after returning to 290 mOsmol kg-1, indicating that osmotic damage occurs during the initial change from isosmotic to hyposmotic media. These osmotic characteristics could be used to determine optimal conditions for cryopreservation of boar spermatozoa.

Animals↗
PubMed1995

Platelet microparticles bind, activate and aggregate neutrophils in vitro.

The interaction of activated platelets with leukocytes are believed to play an important role in ischemic reperfusion injury and other thrombotic conditions. Upon activation, platelets shed platelet microparticles (PMP) and express activation markers CD62P expressed on activated platelets mediates adhesion of platelets to leukocytes, chiefly neutrophils, but little is known of the interaction of PMP isolated from stored platelets or thrombin activated platelets was incubated with leukocytes and binding assessed by flow cytometry. FITC-labeled alpha-CD41 was used to assess platelet material associated with WBC. Like platelets PMP bound preferentially to neutrophils rather than lymphocytes, and exhibited an absolute dependence on the presence of Ca2+. Binding was time-and concentration-dependent, reaching a plateau at 10 min at a ratio of PMP to neutrophils of 150:1. Fluorescence microscopy showed that most of the neutrophils were aggregated into clusters of 5-20 cells. Clustering of neutrophils was not observed to result form interaction with platelets. In these clusters the adherent PMP appeared to serve as bridges between the neutrophil. Addition of EGTA after brief incubation (5-10 min) released most of the bound PMP but if added after > 10 min, only approximately 60% of bound PMP were released. In contrast, nearly all bound platelets were released by EGTA at the same time of incubation. Incubation of neutrophils with PMP gave significantly higher percentage of CD41a(+)neutrophils than did platelets incubated at the same numerical ratio. PMP association with neutrophils was less markedly inhibited by alpha-CD62P (AC1.2) than platelets, but binding of both PMP and activated platelets was inhibited approximately 90% by antisialyl Lewis X. PMP binding to neutrophils induced a significant increase in both CD11b expression and phagocytic activity in a concentration-dependent manner. These findings suggest a possible role for PMP in addition to providing platelet factor 3, specifically, as an activator and mediator of neutrophils in ischemic injury, thrombosis, and inflammation.

Blood Platelets↗
PubMed1995

The effect of collection temperature, cooling rate and warming rate on chilling injury and cryopreservation of mouse spermatozoa.

The experiments presented here identify several factors that affect survival (motility) of cryopreserved mouse spermatozoa after freezing and thawing. Among these factors are: (i) the temperature at which spermatozoa are collected, (ii) the cooling rate to 0 degrees C and (iii) the warming rate from -196 degrees C to ambient. When excised epididymides were cooled to near 0 degrees (1-4 degrees C) and spermatozoa collected and mixed with cryoprotectant at that temperature, motilities after subsequent freezing and thawing were 8-10 times higher than when the spermatozoa were collected from the epididymides at 22 degrees C. In addition, the survival rates of spermatozoa warmed at rates ranging from 150 to 2000 degrees C min-1 were about five times higher than those in suspensions warmed at about 7500 degrees C min-1. The combination of a low collection temperature and the lower warming rates resulted in approximately 50% motility relative to unfrozen controls. Motility was reduced to 6-8% when the collection temperature was 22 degrees C, and to approximately 10% when frozen suspensions of spermatozoa collected in the cold were rapidly warmed from -196 degrees C. When spermatozoa collected at 22 degrees C were abruptly cooled to 0 degrees C, 40-80% of the cells suffered an irreversible loss of motility after warming. In contrast, when spermatozoa were cooled to 0 degrees C at 1 degree C min-1 and warmed (either rapidly or slowly), motilities were similar to those of uncooled controls (75-90%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

[Effect of erythroid enhancer on the expression of beta-globin gene in mice erythroleukemia (MEL) cells].

Our previous works have verified that the beta-globin gene carrying large fragments of erythroid enhancer transferred by retrovirus vector caused the unstable provirus integration and low virus titer in infected cells, but the 36bp enhancer had not this negative effect. In order to circumvent this problem, we inserted the intact beta-globin gene (beta) or partially IVS II deleted beta-globin gene (delta beta) and truncated erythroid enhancer (36bp, 292bp and 341bp) into the N2A retrovirus vector. Recombinants were transfected into psi-2 ecotropic pachaging cells first, then the produced virus were used to infect PA317 amphotropic packaging cells. Virus supernatent from PA317 clonies with high virus titer and intact provirus integration was used to infect MEL cells. RNase protection assay was used to detect the expression of beta-globin gene. Results showed that not only the stable provirus integration and high virus titer of the transferred genes, but also the high levels expression of beta-globin gene carrying 292bp or 341bp erythroid enhancer were got.

Animals↗
PubMed1994

Determination of boar spermatozoa water volume and osmotic response.

Boar spermatozoa water volume and osmotic response were determined by a shapeindependent method for measuring cellular volume, electron paramagnetic resonance (EPR), employing the spin label, tempone, and the broadening agent, potassium chromium oxalate (CrOx). A water volume of 18.4 +/- 1.6 mum(3) (X +/- SD) was obtained for individual boar spermatozoa at 290 milliosmolar (mOsm) which, after correction for the presence of cytoplasmic droplets, yields a boar sperm water volume of 13.0 to 15.0 mum(3). Assuming 59% of the total cell volume is water, the total cell volume of boar spermatozoa is 22.0 to 25.4 mum(3). In addition, the experiment indicated that the relative water volume versus the reciprocal of the external osmolality (Boyle van't Hoff plot) was linear over the range of 210 to 1500 mOsm of sodium chloride (r(2) = 0.996), supporting the hypothesis that boar spermatozoa act as ideal osmometers. A non-zero y axis intercept of 0.23 from the Boyle van't Hoff plot indicated a 23% spin label accessible, but osmotically inactive water component.

Journal Article↗
PubMed1994

Water volume and osmotic behaviour of mouse spermatozoa determined by electron paramagnetic resonance.

Experiments were conducted to determine the water volume and osmotic behaviour of mouse spermatozoa using an electron paramagnetic resonance technique using the spin label tempone, and the broadening agent potassium chromium oxalate. After a swim-up procedure, an average water volume of 43.3 micron3 of individual spermatozoa was obtained at 290 mosmol. If a water compartment of 59% is assumed, the total volume of mouse spermatozoa is 73.4 micron3. A plot of the relative water volume of mouse spermatozoa versus the reciprocal of buffer osmolality (Boyle van't Hoff plot) is linear in the range 250-900 mosmol of sodium chloride solutions (r2 = -.96). The Boyle van't Hoff plot intercept indicates that 13% of the spin-label accessible isotonic water is osmotically inactive.

Animals↗
PubMed1994

Membrane transport properties of mammalian oocytes: a micropipette perfusion technique.

A perfusion technique using micropipette methodology was developed to determine quantitatively the membrane transport properties of mammalian oocytes. This method eliminates modelling ambiguities inherent in microdiffusion, a closely related technology, and should prove to be especially valuable for study of the coupled transport of water and cryoprotectant through mammalian oocytes and embryos. The method is described and evidence given for validity of the method for the simple case of uncoupled flow of water through the mouse oocyte membrane. The zona pellucida of a mouse oocyte was held by a micropipette with an 8-10 microns diameter tip opening and perfused by hyperosmotic media. The kinetic volume change of the cell was videotaped and quantified by image analysis. Experimental data and mathematical modelling were used to determine the hydraulic conductivity of the oocyte membrane (Lp) found to be 1.05, 0.45 and 0.26 microns min-1 atm-1 at 30 degrees C, 22 degrees C and 12 degrees C, respectively. The corresponding activation energy, Ea, for Lp was calculated to be 13.0 kcal mol-1. These values are in agreement with data obtained by other techniques. One of the major advantages of this technique is that the extracellular osmotic condition can be changed readily by perfusing a single cell with a prepared medium. To study the response of the same cell to different osmotic conditions, the old perfusion medium can be removed easily and the cell reperfused with a different medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

[Detection of B-cell clonality in paraffin-embedded bone marrow biopsy specimens by polymerase chain reaction].

In order to estimate the clonality of B-cell lymphoproliferative disorders, polymerase chain reaction (PCR) was used to detect the Ig heavy chain gene rearrangement in DNA extracted from formalin-fixed and paraffin-embedded bone marrow biopsies. 16 out of 19 cases (84%) of B-CLL (n = 9), multiple myeloma (n = 7) and B-non-Hodgkin's lymphoma (n = 3) showed sharp monoclonal bands, while polyclonal smears or no PCR-products were observed in non-neoplastic bone marrow or AML specimens. The results showed that the DNA from paraffin-embedded bone marrow biopsies could be used to detect monoclonal IgH rearrangement by PCR method. This forms the basis for the studies of minimal residual disease or B-ML bone marrow infiltrated diseases.

Biopsy↗