Biomedical subjects

Lan Huang

Publications and source records attributed to Lan Huang.

62 records · Page 4Linked to original sources

PubMed2002

Structure-based experimental confirmation of biochemical function to a methyltransferase, MJ0882, from hyperthermophile Methanococcus jannaschii.

We have determined the three-dimensional (3-D) structure of protein MJ0882, which derives from a hypothetical open reading frame in the genome of the hyperthermophile Methanococcus jannaschii. The 3-D fold of MJ0882 at 1.8 A highly resembles that of a methyltransferase, despite limited sequence similarity to any confirmed methyltransferase. The structure has an S-adenosylmethionine (AdoMet) binding pocket surrounded by motifs with similarities to those commonly found among AdoMet binding proteins. Preliminary biochemical experiments show that MJ0882 specifically binds to AdoMet, which is the essential co-factor for methyltransferases.

Amino Acid Motifs↗

PubMed2002

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.

Amino Acid Sequence↗

PubMed2002

Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry.

The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C(4) reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4.

Acetylation↗

PubMed2002

Deciphering networks of protein interactions at the nuclear pore complex.

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA. More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K(D) = 1.5 micro M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗

PubMed2002

Autologous fixed tumor vaccine: a formulation with cytokine-microparticles for protective immunity against recurrence of human hepatocellular carcinoma.

We developed a tumor vaccine consisting of fixed hepatocellular carcinoma (HCC) cells/tissue fragments, biodegradable microparticles encapsulating granulocyte-macrophage-colony stimulating factor and interleukin-2, and an adjuvant. The vaccine protected 33% of syngeneic mice from HCC cell challenge. The vaccine containing human autologous HCC fragments showed essentially no adverse effect in a phase I/IIa clinical trial and 8/12 patients developed a delayed-type hypersensitivity (DTH) response against the fragments. Although 2 of 4 DTH-response-negative patients had recurrence after curative resection, the DTH-response-positive patients had no recurrence. The time before the first recurrence in the vaccinated patients was significantly longer than that in 24 historical control patients operated in the same department (P < 0.05). This formulation is a promising candidate to prevent recurrence of human HCC.

Animals↗

PubMed2002

[Determination of piceid in commercial Rhizoma Polygoni Cuspidati by HPLC].

OBJECTIVE: To improve a RP-HPLC method of determining piceid in commercial Rhizoma Polygoni Cuspidati. METHOD: HPLC method was used, with C18 column (250 mm x 4.6 mm), a mixture of CH3CN-H2O (20:80) as the mobile phase, and detecting wavelength 303 nm. RESULT: The linear range was 0.0066-0.0792 microgram.microL-1, r = 0.9999 (n = 5). The average recovery rate was 98.5% and RSD was 2.5% (n = 9). CONCLUSION: The method is simple, accurate and reproducible. The contents of piceid in commercial Rhizoma Polygoni Cuspidati from different areas are variable and the average content is 2.23%.

Chromatography, High Pressure Liquid↗

PubMed2002

[The preliminary study on noninvasive detection using NIR diffusion reflectance spectrum for monitoring blood glucose].

The noninvasive detection method using near-infrared diffusion reflectance spectrum for monitoring blood glucose was described. A group of healthy volunteers(2 young male and 2 young female) was tested twenty-six times under changing of both the blood glucose concentration and spectrum region during various states. In order to set up a model, the set was chosen from these samples, the set of prediction was not belong to the set of the model. The results from different selected spectrum regions and reprocessing methods show as follows: 1) In the spectrum region of 9,000-12,000 cm-1 using MIN-MAX normalization reprocessing method to set up a model and predict, the errors of prediction were more than 36 mg.dL-1; the model errors were under 16 mg.dL-1 and predictive errors were less than 25 mg.dL-1 by using smoothing and second derivatives in the same region; in the another region of 4,000-5,000 cm-1 with smoothing and first derivatives both error of model and prediction were more than 25 mg.dL-1. 2) In the region of 4,000-9,000 cm-1 with smoothing and second derivatives, the errors from model were less than 15 mg.dL-1, the errors of prediction were less than 31 mg.dL-1; selecting the spectrum regions of 6,100-7,500 cm-1 and 4,200-4,700 cm-1 by a quantitative software OPU VERSION 3.01 with smoothing, first derivatives and vector normalization, the errors of model were no more than 11 mg.dL-1, the errors of predication were no more than 22 mg.dL-1. 3) Choosing the sample from the subject itself to establish the model in 9,000-12,000 cm-1 with smoothing and second derivatives, the errors of model and predication were less than 15 mg.dL-1 and 11 mg.dL-1 respectively. The results showed that the method was feasible to monitoring the blood glucose concentration by selecting an appropriate spectrum region and reprocessing methods with partial least square algorithm which set up a mathematical model. Excepting the number of sample, selected spectrum region and reprocessing methods, the other influencing factors such as the variant from the subject were discussed also.

Blood Glucose Self-Monitoring↗

PubMedDate not supplied

Monitoring cerebral oxygen saturation during cardiopulmonary bypass using near-infrared spectroscopy: the relationships with body temperature and perfusion rate.

During cardiopulmonary bypass (CPB) because of weak arterial pulsation, near-IR spectroscopy (NIRS) is almost the only available method to monitor cerebral oxygenation noninvasively. Our group develops a NIRS oximeter to monitor regional cerebral oxygenation especially its oxygen saturation (rScO2). To achieve optimal coupling between the sensor and human brain, the distances between the light source and the detectors on it are properly chosen. The oximeter is calibrated by blood gas analysis, and the results indicate that its algorithm is little influenced by either background absorption or overlying tissue. We used it to measure the rScO2 of 15 patients during CPB. It is shown that rScO2 is negatively correlated with body temperature and positively with perfusion rate. There are two critical stages during CPB when rScO2 might be relatively low: one is the low-perfusion-rate stage, the other is the early rewarming stage. During cooling, the changes of total hemoglobin concentration (C(tHb)) compared with its original value is also monitored. It is shown that C(tHb) decreases to a small extent, which may mainly reflect cerebral vasoconstriction induced by cooling. All these results indicate that NIRS can be used to monitor cerebral oxygenation to protect cerebral tissue during CPB.

Adolescent↗