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Biomedical subjects

X Hu

Publications and source records attributed to X Hu.

At least 343 records · Page 19Linked to original sources
PubMed1996

Production of tumor necrosis factor-alpha as a result of glia-T-cell interaction correlates with the pathogenic activity of myelin basic protein-reactive T cells in experimental autoimmune encephalomyelitis.

Tumor necrosis factor-alpha (TNF-alpha) has attracted the greatest attention as a major factor in experimental autoimmune encephalomyelitis (EAE) pathogenesis. We compared rats undergoing EAE with manipulated but healthy animals by examining TNF-alpha gene expression in cells recovered from the brain. We used reverse transcriptase-polymerase chain reaction (RT-PCR) as a sensitive assay for detection and Northern blot hybridization as a reliable quantitative assay of TNF-alpha mRNA. TNF-alpha gene expression was consistently detected in rats immunized with myelin basic protein (MBP) emulsified in complete Freund adjuvant (CFA), but not in rats immunized with MBP emulsified in incomplete Freund adjuvant (IFA), which does not induce EAE. Similarly, brain-derived cells from rats injected with cloned encephalitogenic T cells contained increased amounts of TNF-alpha mRNA compared with rats injected with nonencephalitogenic T cell clones similar in antigen specificity and in vitro lymphokine-producing capacity. Considering that the differing pathogenic capacity of MBP-reactive T cells might result from differing patterns of interaction with glia, we examined the impact of T-cell-glia interaction in vitro on cytokine gene expression in both cell types. Glial components were efficient in inducing TNF-alpha expression in T cells; T cells and T-cell-derived cytokines could elicit expression of several lymphokine genes in glial cells. Comparison of RT-PCR and blot hybridization assays, however, suggested that cytokine expression was much more efficient, on a per cell basis, in T cells than in glia. TNF-alpha was shown to have direct cytotoxic effect on glial cells, which was greatly enhanced by small amounts of interferon-gamma (IFN-gamma).

Animals↗
PubMed1996

Novel anti-carcinogenic activity of an organosulfide from garlic: inhibition of H-RAS oncogene transformed tumor growth in vivo by diallyl disulfide is associated with inhibition of p21H-ras processing.

In this study, we report a novel anticarcinogenic activity of an organosulfur compound from garlic, diallyl disulfide (DADS). DADS treatment significantly inhibited the growth of H-ras oncogene transformed tumors in nude mice. As compared to controls, the appearance of tumors was also delayed markedly by oral administration of DADS. The inhibition of tumor growth by DADS treatment correlated with the inhibition of p21H-ras membrane association in the tumor tissue. The levels of membrane associated p21H-ras were markedly lower in the tumor tissues of DADS treated mice as compared to controls. An opposite trend, however, was evident for cytosolic p21H-ras. Furthermore, DADS treatment resulted in a significant inhibition of hepatic as well as tumoral 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. These results indicate that DADS suppresses the growth of H-ras oncogene transformed tumors in nude mice by inhibiting the membrane association of tumoral p21H-ras.

Allyl Compounds↗
PubMed1996

Lack of a role of glutathione in cellular nonenzymatic activation of BMS-181174, a novel analogue of mitomycin C.

Recent studies, using a cell-free system, have suggested that thiol-dependent nonenzymatic bioactivation may be responsible for the superior antitumor activity of the mitomycin C analogue BMS-181174 [N-7-[2-(4-nitrophenyldithio)ethyl]mitomycin C] when compared to the parent compound. If operational in tumor cells, this pathway could have enormous clinical implications since tumor cell resistance to a variety of anticancer agents is often associated with increased glutathione (GSH) levels and BMS-181174 may be used to reverse this mechanism of resistance. The present study was undertaken to determine the role of GSH in cellular activation of BMS-181174 using a pair of well-characterized human bladder cancer cells (J82 and SCaBER) as a model. A 20-h pretreatment of J82 and SCaBER cells with a nontoxic concentration of D,L-buthionine-S,R-sulfoximine (BSO) caused about 80-88% reduction in cellular GSH levels. Surprisingly, the sensitivity of both cells to BMS-181174 was increased, not reduced, by BSO-induced GSH depletion. On the other hand, the cytotoxicity of BMS-181174 was significantly reduced in both cells by a 4-h pretreatment with 1 mM GSH. Like BSO, a 4-h pretreatment with another thiol compound (cysteine) resulted in a statistically significant sensitization of both cells to BMS-181174. Cellular GSH levels were not affected in either of the cell lines by pretreatment with GSH or cysteine. In conclusion, the results or the present study argue against a role of GSH in cellular nonenzymatic activation of BMS-181174 in J82 and SCaBER cells.

Antineoplastic Agents, Alkylating↗
PubMed1996

Phase II clinical trial with 5-fluorouracil, recombinant interferon-alpha-2b, and cisplatin for patients with metastatic or regionally advanced carcinoma of the esophagus.

BACKGROUND: Recombinant interferon-alpha (IFN) augments the cytotoxicity of both 5-fluorouracil (5-FU) and cisplatin in vitro. A phase II study of 5-FU and IFN resulted in response rates of 25-27% in patients with metastatic esophageal carcinoma. METHODS: A Phase II trial was initiated to determine the clinical utility of a three-drug combination (FIP) in patients with regionally advanced or metastatic esophageal carcinoma. Eligibility included biopsy-proven Stage III or IV squamous cell carcinoma or adenocarcinoma of the esophagus with no prior chemotherapy, adequate performance status, nutritional status, bone marrow, hepatic and renal function, and signed informed consent. Patients were treated in the exact sequence of IFN==>cisplatin==>5-FU. Patients received 5-FU, 750 mg/m2/day for 5 days followed by weekly bolus therapy at the same dose; cisplatin, 100 mg/m2 on Day 1, followed by weekly therapy, 25 mg/m2 over the course of 1 hour; and IFN, 10 MU subcutaneously 3 times/week beginning on Day 1. All patients received sargramostim (granulocyte-macrophage colony-stimulating factor, Escherichia coli-derived), 5 micrograms/kg subcutaneously 5 times/week. No patients received radiotherapy. RESULTS: Twenty-four patients were enrolled; 23 were eligible, and 1 was excluded on pathology review (patient was found to have a leiomyoblastoma). The demographics of the population were: median age, 63 years (range, 43-73 years); 18 male patients; squamous cell carcinoma: adenocarcinoma ratio, 22:1, and Stage III:IV ratio, 10:13. Grade 3-4 National Cancer Institute Common Toxicity Criteria toxicities included: leukopenia (13), thrombocytopenia (14), and infection (9). Grade 3 diarrhea, mucositis, and vomiting occurred in 6 patients, 4 patients, and 1 patient, respectively. There were two instances of sudden death, likely related to tumor progression. Major responses occurred in 15 of 23 patients (65%; 95% confidence interval, 43%, 85%) (1 complete response, 14 partial responses). The median survival was 8.6 months; with a median follow-up of 26 months, estimated 30-month survival was 31%. CONCLUSIONS: This regimen, although moderately toxic, has substantial activity in metastatic and regionally advanced squamous cell carcinoma of the esophagus. Further investigations should be conducted to determine the role of IFN in the treatment of esophageal carcinoma.

Adenocarcinoma↗
PubMed1996

Enhancement of cytolytic T lymphocyte precursor frequency in melanoma patients following immunization with the MAGE-1 peptide loaded antigen presenting cell-based vaccine.

Identification of human melanoma-associated peptide antigens for CTLs has opened unprecedented opportunities for active specific immunotherapy for melanoma with synthetic peptide. We have shown that immunization with a MAGE-1 gene encoded nonapeptide (EADPT-GHSY)-pulsed autologous antigen presenting cell-based vaccine induces autologous melanoma-reactive and peptide-specific CTL response, in situ, at the vaccination site and at distant tumor deposits in patients who are HLA-A1+ and whose melanoma cells express the MAGE-1 mRNA. Here, we show that such immunization is also capable of increasing the frequency of autologous melanoma-reactive CTL precursors in the circulation. We further show that in vitro stimulation of the postimmunization peripheral blood lymphocytes with the MAGE-1 nonapeptide-loaded antigen presenting cell and interleukin-2 leads to significant expansion of peptide-specific and autologous melanoma-reactive CTL response.

Amino Acid Sequence↗
PubMed1996

The crystal structure of the light-harvesting complex II (B800-850) from Rhodospirillum molischianum.

BACKGROUND: The light-harvesting complexes II (LH-2s) are integral membrane proteins that form ring-like structures, oligomers of alpha beta-heterodimers, in the photosynthetic membranes of purple bacteria. They contain a large number of chromophores organized optimally for light absorption and rapid light energy migration. Recently, the structure of the nonameric LH-2 of Rhodopseudomonas acidophila has been determined; we report here the crystal structure of the octameric LH-2 from Rhodospirillum molischianum. The unveiling of similarities and differences in the architecture of these proteins may provide valuable insight into the efficient energy transfer mechanisms of bacterial photosynthesis. RESULTS: The crystal structure of LH-2 from Rs. molischianum has been determined by molecular replacement at 2.4 A resolution using X-ray diffraction. The crystal structure displays two concentric cylinders of sixteen membrane-spanning helical subunits, containing two rings of bacteriochlorophyll-a (BChl-a) molecules. One ring comprises sixteen B850 BChl-as perpendicular to the membrane plane and the other eight B800 BChl-as that are nearly parallel to the membrane plane; eight membrane-spanning lycopenes (the major carotenoid in this complex) stretch out between the B800 and B850 BChl-as. The B800 BChl-as exhibit a different ligation from that of Rps. acidophila (aspartate is the Mg ligand as opposed to formyl-methionine in Rps. acidophila). CONCLUSIONS: The light-harvesting complexes from different bacteria assume various ring sizes. In LH-2 of Rs. molischianum, the Qy transition dipole moments of neighbouring B850 and B800 BChl-as are nearly parallel to each other, that is, they are optimally aligned for Föster exciton transfer. Dexter energy transfer between these chlorophylls is also possible through interactions mediated by lycopenes and B850 BChl-a phytyl tails; the B800 BChl-a and one of the two B850 BChl-as associated with each heterodimeric unit are in van der Waals distance to a lycopene, such that singlet and triplet energy transfer between lycopene and the BChl-as can occur by the Dexter mechanism. The ring structure of the B850 BChl-as is optimal for light energy transfer in that it samples all spatial absorption and emission characteristics and places all oscillator strength into energetically low lying, thermally accessible exciton states.

Amino Acid Sequence↗
PubMed1996

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring.

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1-20 ng/ml for urine and 2.5-250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.

Analgesics, Opioid↗
PubMed1996

Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli.

Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.

Amino Acid Sequence↗
PubMed1996

A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain.

Calmodulin, a ubiquitous calcium-binding protein, regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. Here, we report isolation of a cDNA encoding a novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe. Calcium-dependent binding of the cDNA-encoded protein to calmodulin is confirmed by 35S-labeled calmodulin. Sequence analysis of a full-length cDNA indicates that it codes for a protein of 1261 amino acids. The predicted amino acid sequence of the KCBP has a domain of about 340 amino acids in the COOH terminus that shows significant sequence similarity with the motor domain of kinesin heavy chains and kinesin-like proteins and contains ATP and microtubule binding sites typical of these proteins. Outside the motor domain, the KCBP has no sequence similarity with any of the known kinesins, but contains a globular domain in the NH2 terminus and a putative coiled-coil region in the middle. By analyzing the calmodulin binding activity of truncated proteins expressed in Escherichia coli, the calmodulin binding region is mapped to a stretch of about 50 amino acid residues in the COOH terminus region of the protein. Using a synthetic peptide, the calmodulin binding domain is further narrowed down to a 23-amino acid stretch. The synthetic peptide binds to calmodulin with high affinity in a calcium-dependent manner as judged by electrophoretic mobility shift assay of calmodulin-peptide complex. The KCBP is coded by a single gene and is highly expressed in developing flowers and suspension cultured cells. Although many kinesin heavy chains and kinesin-like proteins have been extensively characterized at the biochemical and molecular level in evolutionarily distant organisms, none of them is known to bind calmodulin. The plant kinesin-like protein with a calmodulin binding domain and a unique amino-terminal region is a new member of the kinesin superfamily. The presence of a calmodulin-binding motif in a kinesin heavy chain-like protein suggests a role for calcium and calmodulin in kinesin-driven motor function(s) in plants.

Amino Acid Sequence↗
PubMed1996

Development of a screening set for new (CAG/CTG)n dynamic mutations.

The expansion of a (CAG/CTG)n triplet repeat has been found to be associated with at least seven genetic diseases, suggesting that this mechanism of disease may be fairly common. To accelerate the discovery of new loci containing (CAG/CTG)n triplet expansions, we have isolated numerous genomic clones containing this class of repeats. We have developed 338 sequence-tagged sites (STSs) containing (CAG/CTG)n repeat sequences. Two hundred ninety-nine STSs were unambiguously assigned to chromosomes, and 89 of the total were assigned to YACs. The 141 STSs that were developed based on (CAG/CTG)n repeats of at least seven units were genotyped on four reference CEPH individuals to estimate their polymorphic quality.

Base Sequence↗
PubMed1996

Differential displaying of mRNAs from the atrioventricular region of developing chicken hearts at stages 15 and 21.

In an effort to isolate novel genes that may be involved in the development of the cardiac cushions and then the formation of cardiac valves and septa, we utilized the differential mRNA display method in conjunction with the whole-mount in situ hybridization. The total RNAs used to differentially display were prepared from atrioventricular (AV) canal regions of stage 15 and stage 21 chicken hearts because critical events known to be important for the AV valve and septum formation occur during this period of the development. We have successfully obtained 14 potential candidate genes. Three examples, 15H16 (phospholamban), E13 (skeletal alpha-tropomyosin) and 21C (a novel gene), are discussed here. Levels of mRNA expression in developing hearts were determined by Northern blot analysis and their expression patterns were revealed and compared using whole-mount in situ hybridization. Both phospholamban and skeletal alpha-tropomyosin messages in the myocardium of the AV canal region showed significant decrease during this period of the development. The 21C differential display product detects a novel 9.5 Kb message whose expression is cardiac-specific at early stages of development. The expression level of the 21C gene appeared to be increased from stage 15 to stages 21 and 25 as determined by both Northern blot analysis and in situ hybridization. From these data, we demonstrate that the differential display method together with the whole-mount in situ hybridization could be an effective means for the isolation of novel and differentially expressed genes.

Animals↗
PubMed1996

Retrospective estimation and correction of physiological artifacts in fMRI by direct extraction of physiological activity from MR data.

A physiological artifact reduction method based on extracting respiratory motion and cardiac pulsation directly from functional MR data is described. In fast low angle shot (FLASH), respiratory cycles are derived utilizing the phase of the center of a navigator echo, in echo-planar imaging (EPI) from the phase of the center k-space point. Cardiac cycles are determined from projections obtained from the navigator echo (FLASH) and the center k-space line (EPI). Because direct extraction of physiological parameters eliminates the need for external monitoring, the method can be more readily implemented. Experimental results illustrate that the technique provides effective compensation for physiology-related signal fluctuations in functional MRI and performs as well as the retrospective technique using external physiological monitoring.

Artifacts↗
PubMed1996

Fast interleaved echo-planar imaging with navigator: high resolution anatomic and functional images at 4 Tesla.

Echo-planar imaging (EPI) is sensitive to magnetic field inhomogeneities, which lead to signal loss and geometric distortions of the image. Magnetic field inhomogeneities induced by susceptibility differences, as encountered in the human body, increase with the magnetic field strength, thus, complicating implementation of high resolution EPI techniques on high magnetic field systems. These problems were overcome by using a fast multishot high resolution EPI method that uses variable flip angles, center-out k-space sampling, and navigator echoes. This approach maximizes signal-to-noise ratio, reduces flow artifacts, and permits correction of intersegment amplitude and phase variations, providing high spatial and temporal resolution. This scheme can be implemented with a single magnetization preparation for contrast that precedes the segments. The utility of this ultrafast segmented EPI technique with navigator is demonstrated for anatomic and functional imaging studies on the human brain at 4 T.

Artifacts↗
PubMed1996

Artifact reduction in EPI with phase-encoded reference scan.

Echo-planar imaging is becoming widely used for various applications. However, because of inherent limitations, images obtained with EPI are often degraded by artifacts arising from the inconsistency between the odd and even echoes in the acquired data. Several correction techniques, including those based on reference measurement, have been previously reported. In this paper, a correction method based on phase-encoded reference measurement is described. This technique is demonstrated with experimental data obtained at 4 Tesla and compared with techniques that use nonphase-encoded reference scan. Results from experimental studies indicate that the present approach reduces the ghost intensity by a factor of 2-3.

Algorithms↗