Biomedical subjects

H He

Publications and source records attributed to H He.

At least 145 records · Page 8Linked to original sources

PubMed1998

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C.

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1998

Inhibition by the JAK/STAT pathway of IFNgamma- and LPS-stimulated nitric oxide synthase induction in vascular smooth muscle cells.

Inducible nitric oxide synthase (iNOS) is induced in many cell types by cytokines and lipopolysaccharide (LPS). Cytokine signal transduction is believed to be mediated primarily through the JAK/STAT pathway. We therefore examined the effects of a JAK2-specific inhibitor, an antisense oligonucleotide to JAK2, and electroporation of neutralizing anti-STAT1 and anti-STAT3 antibodies on IFNgamma- and LPS-stimulated induction of iNOS in vascular smooth muscle cells. Unexpectedly, we found that the JAK/STAT pathway suppresses IFNgamma- and LPS-stimulated iNOS induction in these cells. In contrast, the JAK/STAT pathway appears to have a positive role in iNOS induction in RAW 264.7 macrophages.

Animals↗

PubMed1998

c-Fos degradation by the proteasome. An early, Bcl-2-regulated step in apoptosis.

c-Fos is a transcription factor that promotes cell growth, differentiation, and transformation. We found that c-Fos was degraded when WEHI7.2 mouse lymphoma cells were induced to undergo apoptosis with the calcium ATPase inhibitor, thapsigargin, or the glucocorticoid hormone, dexamethasone. The degradation of c-Fos preceded caspase-3 activation and apoptotic nuclear chromatin condensation and was inhibited by the proteasome inhibitors MG132, N-acetyl-leucyl-leucyl-norleucinal, and lactacystin. Stable transfection of WEHI7.2 cells with a mutant form of c-Fos that was not degraded by the proteasome inhibited apoptosis. Also, overexpression of Bcl-2 in WEHI7.2 cells blocked c-Fos degradation and inhibited apoptosis. The results indicate that proteasome-mediated degradation of c-Fos is an early, Bcl-2-regulated step in apoptosis induction by thapsigargin and dexamethasone. These findings suggest that c-Fos may have a protective action that is eliminated by proteasome-mediated degradation and preserved by Bcl-2.

Amino Acid Sequence↗

PubMed1998

The Raf-MEK-ERK cascade represents a common pathway for alteration of intracellular calcium by Ras and protein kinase C in cardiac myocytes.

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating phorbol ester, phorbol 12-myristate 13-acetate, also prolonged the contractile [Ca2+]i transients. Cotransfection with dnMEK inhibited the effects of Ha-RasV12, Raf, and phorbol 12-myristate 13-acetate on [Ca2+]i. The effects of Ha-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function.

Animals↗

PubMed1998

On-the-fly fluorescence lifetime detection of dye-labeled DNA primers for multiplex analysis.

Mixtures of dye-labeled, M13-forward DNA primers were separated by capillary gel electrophoresis and detected on-the-fly, using fluorescence lifetime measurements, to evaluate four-decay detection for multiplex DNA sequencing. Three different four-dye systems were used, two that were excited at 488 nm and one that was excited at 514 nm. Each dye-labeled primer was identified on the basis of the lifetime of the conjugated dye using nonlinear least squares or the maximum entropy method to analyze the lifetime data. Overlapping electrophoretic peaks were generated by making multiple injections of mixtures of the dye-labeled primers. The overlapping peaks were resolved by fitting the data to two-, three- or four-component lifetime models used in nonlinear least-squares analysis in which each lifetime component was fixed to the predetermined lifetime of the corresponding dye-labeled primer. In two of the dye systems, the lifetimes of the four dye-labeled primers were sufficiently different to allow peak resolution. In the other dye system, addition of 10% DMSO to the run buffer changed the lifetime of one dye-labeled primer, allowing it to be resolved from another dye-labeled primer with similar lifetime.

DNA Primers↗

PubMed1998

Characterization of a novel Bax-associated protein expressed in hemopoietic tissues and regulated during thymocyte apoptosis.

Members of the Bcl-2 protein family have been implicated as critical intracellular regulators of apoptosis. Most studies of this protein family have utilized transformed and/or transfected cell lines expressing high levels of these proteins. In the current study, we have analyzed normal murine lymphoid cells and tissues and have detected a previously unreported protein of approximately 16 kDa recognized by an anti-Bax Ab. This 16-kDa protein is abundant in hemopoietic tissues of both wild-type and Bax knock-out mice, it can heterodimerize with Bax in normal lymphocytes, and it is dramatically down-modulated in thymocytes in response to apoptotic stimuli. These results suggest that this protein may have antiapoptotic activity and may participate in the regulation of apoptosis in normal lymphocytes.

Animals↗

PubMed1998

Down-modulation of a novel Bax-associated protein during apoptosis in normal mature B lymphocytes.

We have recently characterized a novel 16-kDa Bax-associated protein. In this study, we investigate the regulation of this protein's expression during in vitro induction of apoptosis in mature splenic B cells. A panel of biochemically distinct apoptotic stimuli induced the dramatic down-modulation of the 16-kDa protein in B cells; this down-modulation was rapid, and did not require DNA fragmentation. Reciprocally, stimuli that induced protection from apoptosis prevented down-modulation of the 16-kDa protein. These regulatory effects were specific, since Bcl-2 and Bax protein levels were not similarly modulated. Stimuli that reduce expression of the 16-kDa protein may therefore act indirectly to increase the proapoptotic activity of Bax, perhaps by altering Bax binding to other cellular proteins.

Animals↗

PubMed1998

Site-specific insertion of a SINE-like element, Cp1, into centromeric tandem repeats from Chironomus pallidivittatus.

A SINE-like dispersed element, Cp1, from the dipteran Chironomus pallidivittatus was found to show site-specific insertion into two different centromeric tandem repeats. The insertions result in identical target site duplications of nine base-pairs. In contrast, extracentromeric Cp1 elements, which are polymorphic and degenerate, are previously known to be surrounded by different target site duplications. The intracentromeric Cp1 is uniform in structure and contains a single pol III unit, upstream of which 87 bp arms of a palindrome surround a 103 bp unique sequence. The numbers of Cp1 elements per centromere were determined in microdissected material and were found to be in the range of five to ten units per centromere. The well-defined insertion properties, correlated to chromosomal localization, suggest that Cp1 is likely to be a component of importance for the centromere. Similarities of Cp1 and its parts to functionally identified centromeres in Saccharomyces cerevisiae and Schizosaccharomyces pombe are discussed.

Animals↗

PubMed1998

pH-dependent fluoride transport in intestinal brush border membrane vesicles.

Fluoride (F) absorption from the rat stomach and urinary bladder, hamster cheek pouch, and the renal tubules of several species are pH gradient-dependent. These observations led to the hypothesis that F crosses these epithelia in the form of the undissociated acid, HF. Several recent reports, however, have provided evidence that F absorption from the rat small intestine is insensitive to the lumenal pH. We report here our evidence that F uptake by rabbit intestinal brush border membrane vesicles (BBMV) occurred rapidly and with an overshoot only in the presence of an inward-directed proton gradient. In the absence of a proton gradient or in the presence of an outward-directed gradient, F uptake was slow and without an overshoot. In the presence of an inward-directed proton gradient, F uptake was partially inhibited by DIDS and DEP but not by diBAC. PCMBS inhibited F uptake by up to 83% in a dose-response manner. DiBAC appeared to reduce intravesicular pH slightly but the other reagents had no effect. When the uptake buffer contained chloride or nitrate, F uptake was partially inhibited compared to the mannitol or gluconate controls. It was concluded that F transport across the rabbit intestinal BBMV occurs via a carrier-mediated process which may involve cotransport of F with H+ or exchange of F with OH-. The inhibitory effects of DIDS, DEP and PCMBS may occur by affecting this carrier-mediated transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

In vitro thymocyte maturation is associated with reduced cellular susceptibility to Fas-mediated apoptosis.

We have developed a novel system in which the susceptibility of murine thymocytes to Fas-mediated apoptosis can be modulated. Thymocyte susceptibility to Fas decreases under in vitro culture conditions that promote aspects of thymocyte maturation. The hyporesponsive state is specific for the Fas pathway, since cellular susceptibility to other apoptotic stimuli is not reduced. Hyporesponsiveness is not associated with alterations in the thymocyte subset distribution, decreased expression of full-length Fas protein, or alterations in FADD, Bcl-2, or Bcl-XL expression. Hyporesponsiveness is overcome by increasing the strength of the Fas cross-linking stimulus, leading us to propose that reduced thymocyte susceptibility to apoptosis results from altered Fas signaling. The block in Fas signaling resides proximal to ceramide generation, since Fas-hyporesponsive thymocytes are susceptible to ceramide-induced apoptosis. Further characterization of Fas signaling in these in vitro cultured thymocytes may facilitate the identification of factors regulating the susceptibility of wild-type lymphocytes to Fas.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Apoptosis induction by the glucocorticoid hormone dexamethasone and the calcium-ATPase inhibitor thapsigargin involves Bc1-2 regulated caspase activation.

The requirement for caspases (ICE-like proteases) were investigated in mediating apoptosis of WEHI7.2 mouse lymphoma cells in response to two death inducers with different mechanisms of action, the glucocorticoid hormone dexamethasone (DX) and the calcium-ATPase inhibitor thapsigargin (TG). Apoptosis induction by these agents followed different kinetics, and was closely correlated with in vivo activation of caspase-3 (CPP32/Yama/Apopain) and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP). Caspase activation and PARP cleavage were inhibited by Bcl-2 overexpression. Cell extracts from DX- and TG-treated cells cleaved the in vitro synthesized baculovirus p35 ICE-like protease target, producing 25 and 10 kDa fragments. p35 cleavage was inhibited by mutating the active site aspartic acid to alanine, and by a panel of protease inhibitors that inhibit caspase-3-like proteases, including iodoacetamide, N-ethylmaleimide, and Ac-DEVD-cho. Treatment of cells in vivo with two cell permeant peptide fluoromethylketone inhibitors of caspase activity, Z-VAD-fmk and Z-DEVD-fmk, inhibited DX- and TG-induced apoptotic nuclear changes and maintained plasma membrane integrity, whereas the cathepsin inhibitor, Z-FA-fmk, and two calpain inhibitors failed to inhibit apoptosis. An unexpected observation was that due to the delayed time course of DX-induced apoptosis, optimal preservation of plasma membrane integrity was achieved by adding caspase inhibitors beginning 8 h after DX addition. In summary, the findings indicate that two diverse apoptosis-inducing signals converge into a common Bcl-2-regulated pathway that leads to caspase activation and apoptosis.

Animals↗

PubMed1998

Simultaneous determination of codeine and its seven metabolites in plasma and urine by high-performance liquid chromatography with ultraviolet and electrochemical detection.

A sensitive and selective high-performance liquid chromatography method has been developed for the measurement of codeine and its seven metabolites, norcodeine, morphine, normorphine, codeine-6-glucuronide, morphine-6-glucuronide, morphine-3-glucuronide and norcodeine glucuronide, in plasma and urine. The compounds were recovered from plasma and urine using solid-phase extraction with C18 cartridges and separated on a reversed-phase C8 column with a mobile phase consisting of 77% buffer (5 mM sodium phosphate monobasic and 0.70 mM sodium dodecyl sulfate, pH 2.35) and 23% acetonitrile. Codeine, norcodeine, codeine-6-glucuronide, norcodeine glucuronide and morphine-3-glucuronide were detected by ultraviolet detection at 214 nm, with a detection limit of 0.02 nmol/ml for each compound in plasma. Morphine-6-glucuronide, normorphine and morphine were monitored by electrochemical detection at 350 mV, with a detection limit of 0.003 nmol/ml for each compound in plasma. The assay showed good reproducibility and accuracy using external standardization. The recovery and inter-day variation for all compounds in plasma samples were 63.40-77.90% and 3.49-16.77% (R.S.D.) and while in urine were 64.98-90.13% and 2.93-9.96% (R.S.D.), respectively.

Chromatography, High Pressure Liquid↗

PubMed1998

Selective induction of Na,K-ATPase alpha3 subunit mRNA abundance in cardiac myocytes by retinoic acid.

Retinoic acid (RA) is a high affinity ligand for a nuclear receptor which regulates transcription in target cells. Specific effects of RA on cardiac development and myocardial cell hypertrophy have been demonstrated; however, little information exists concerning RA-mediated regulation of cardiac genes. This study was initiated to investigate whether RA regulates Na,K-ATPase subunit gene expression in primary cultures of neonatal rat cardiac myocytes. Northern blot analyses demonstrated that NA, K-ATPase alpha3 subunit mRNA content was stimulated three-fold by RA. The effect of RA on alpha3 subunit gene expression was selective as RA treatment had no effect on either Na,K-ATPase alpha1, alpha2 or beta1 subunit mRNAs. A stimulatory effect of RA on Na,K-ATPase alpha3 gene transcription was not evident in either transient transfection or nuclear run-on studies, suggesting that augmentation of alpha3 mRNA content by RA was due to a post-transcriptional mechanism. Finally, RA diminished the magnitude of the thyroid hormone (T3)-mediated increase in Na,K-ATPase beta1 subunit mRNA, while RA had no effect on the stimulation of alpha3 mRNA content by T3.

Animals↗

PubMed1998

Study on amino acid composition of HSP70 and the level of plasma free amino acids of workers with long-term exposure to harmful factors.

In order to know the practical value of heat stress protein 70 (HSP70) and to know the changes of plasma free amino acids of workers with the induction of HSP70 by harmful factors, the amino acid composition of major HSP, HSP70 purified from the heated cultured human leukemia cancer cell line K562, rabbit liver, rat liver and heart, and mouse liver with two-step procedures of DE52-cellulose ion exchange chromatography and affinity chromatography on ATP-agarose was examined. The level of plasma free amino acids of workers with long-term exposure to heat, carbon monoxide and the combined effect of both heat and carbon monoxide was also investigated. The results showed that the three richest amino acids in HSP70 of all origins were Gly, Glu and Asp, except that of rat heart which was rich in Gly, Phe and Glu. Additionally, Lys, Val, Leu and Ala were also found very rich in HSP70 of all origins. Compared with controls, the most of plasma free amino acids tended to increase and free methionine and tryptophan were increased significantly (P < 0.05) after a long-term exposure to heat, carbon monoxide, and both. These findings suggested that further studies need to be done to find the substances or drugs which induce the synthesis of HSP70 and reduce the inhibition of synthesis of normal proteins for the purpose of protecting people with exposure to harmful factors against the damage of the factors.

Amino Acids↗

PubMed1998

Centromere 3 specific tandem repeat from Chironomus pallidivittatus.

A 155-bp tandem repeat was previously reported to be present in all centromeric regions of the dipteran Chironomus pallidivittatus. We have now isolated a second centromere specific tandem repeat, 375 bp long. Two blocks were found of the new unit, differing in size, probably representing allelic forms. The repeat is present only in chromosome 3, bordering 155-bp repeat arrays. There are about 100 repeats per genome, compared to 1300 units for the 155-bp repeat. The two units contain an identical 9-bp sequence which can form target-site duplications flanking a short mobile element, Cp1. An inversion within the tandem array was isolated, the breakpoint of which is within the 9-bp target sequence. Another short shared motif, 10-bp long, is also present at the insertion site for a mobile element. The two repeat units are similar in having long regions with more than 80% AT and an overall high AT content.

Animals↗

PubMed1998

Analysis of T-cell activation after bronchial allergen challenge in patients with atopic asthma.

BACKGROUND: T helper cells are a heterogeneous group of cells that have phenotypic and functional differences. Activated T helper cells have been found in peripheral blood after allergen challenge of subjects with atopic asthma, but the phenotypes of specific T helper subpopulation involved remains to be identified. OBJECTIVE: To characterize the T cell activation markers that may be regulated by allergens, we analyzed peripheral blood lymphocytes obtained before and after allergen challenge from subjects with atopic asthma. METHODS: We analyzed the distribution of the cell surface activation markers, interleukin 2 receptor (IL-2R) and major histocompatibility complex class II antigens (MHC II) among T helper subpopulations classified as naive (CD45RA) or memory (CD45RO) phenotypes. Nine adult subjects with atopic asthma underwent bronchoprovacative allergen inhalation and isocapnic cold air hyperventilation (ISH) challenge followed by serial spirometry. Peripheral blood mononuclear cells (PBMC) were isolated at baseline and 2 and 24 hours after challenge. Four-color flow cytometry was used to analyze the expression and distribution in vivo of IL-2R and MHC II activation markers on naive and memory T cell subsets after challenge. RESULTS: At 2 and 24 hours after allergen challenge, there was a significant increase in the CD45RO+IL-2R+ T helper cells compared with baseline (mean +/- SE, baseline, 12.5% +/- 1% versus 2 hours, 18.1% +/- 1% and 24 hours, 17.8% +/- 2%, p < 0.025). MHC II expression was not significantly increased after challenge on naive and memory T helper cells and coexpression of IL-2R and MHC II was only found in a small proportion of CD45RO+ T helper cells (2.7% +/- 1%). No changes of IL-2R or MHC II expression on T helper subsets were observed after ISH challenge in the same patients. We also found that 31% to 46% of T helper cells coexpress CD45RA and CD45RO simultaneously, and upregulation of IL-2-R and MHC II expression occurs only on those T helper cells that express CD45RO. CONCLUSIONS: We have found that T helper cells express both CD45RA and CD45RO isoforms, which suggests the existence of a transitional phenotype among naive and memory T helper cells in peripheral blood. In subjects with atopic asthma, our in vivo analysis characterizes two populations of activated memory T helper cells based on the expression of IL-2R or MHC II surface molecules after allergen challenge.

Adult↗

PubMed1998

23Na NMR studies of Na-DNA in the solid state.

23Na MAS, CP/MAS and quadrupole nutation NMR spectra and the 23Na spin-lattice relaxation times in Na-DNA with and without competing species (Mg2+, ethidium bromide and [Ru(phen)3]2+) reveal the presence of two sodium sites with different NMR parameters. While in the presence of Mg2+, sodium resides far from the surface of the DNA molecule, ethidium bromide and [Ru(phen)3]2+ displace sodium closer towards the surface.

Animals↗

PubMed1998

Role of phosphatidylinositol 4,5-bisphosphate in Ras/Rac-induced disruption of the cortactin-actomyosin II complex and malignant transformation.

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

3T3 Cells↗