Biomedical subjects

Chao Zhang

Publications and source records attributed to Chao Zhang.

At least 91 records · Page 5Linked to original sources

PubMed2003

Cutting edge: a chemical genetic system for the analysis of kinases regulating T cell development.

To understand the regulatory activities of kinases in vivo requires their study across a biologically relevant window of activity. To this end, ATP analog-sensitive kinase alleles (ASKAs) specifically sensitive to a competitive inhibitor have been developed. This article tests whether ASKA technology can be applied to complex immunological systems, such as lymphoid development. The results show that when applied to reaggregate thymic organ culture, novel p56(Lck) ASKAs readily expose a dose-dependent correlation of thymocyte development with a range of p56(Lck) activity. By regulating kinase activity, rather than amounts of RNA or protein, ASKA technology offers a general means for assessing the quantitative contributions to immunology of numerous kinases emerging from genomics analyses. It can obviate the generation of multiple lines of mice expressing different levels of kinase transgenes and should permit specific biological effects to be associated with defined biochemical activities.

Adenosine Triphosphate↗

PubMed2003

Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2.

Meiosis is thought to require the protein kinase Ime2 early for DNA replication and the cyclin-dependent kinase Cdc28 late for chromosome segregation. To elucidate the roles of these kinases, we inhibited their activities early and late using conditional mutants that are sensitive to chemical inhibitors. Our studies reveal that both Cdc28 and Ime2 have critical roles in meiotic S phase and M phase. Early inhibition of analog-sensitive cdc28-as1 blocked DNA replication, revealing a previously undetected role for Cdc28. Yet Cdc28 was dispensable for one of its functions in the mitotic cell cycle, degradation of Sic1. Late addition of inhibitor to ime2-as1 revealed unexpected roles of Ime2 in the initiation and execution of chromosome segregation. The requirement of Ime2 for M phase is partially explained by its stimulation of the key meiotic transcription factor Ndt80, which is needed in turn for high Cdc28 activity. In accordance with a late role for Ime2, we observed an increase in its activity during M phase that depended on Cdc28 and Ndt80. We speculate that several unique features of the meiotic cell division reflect a division of labor and regulatory coordination between Ime2 and Cdc28.

CDC28 Protein Kinase, S cerevisiae↗

PubMed2003

Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain.

By integrating convergent protein engineering and rational inhibitor design, we have developed an in vivo conditional protein knockout andor manipulation technology. This method is based on the creation of a specific interaction interface between a modified protein domain and sensitized inhibitors. By introducing this system into genetically modified mice, we can readily manipulate the activity of a targeted protein, such as alpha-Ca(2+)calmodulin-dependent protein kinase II (alphaCAMKII), on the time scale of minutes in specific brain subregions of freely behaving mice. With this inducible and region-specific protein knockout technique, we analyzed the temporal stages of memory consolidation process and revealed the first postlearning week as the critical time window during which a precise level of CaMKII reactivation is essential for the consolidation of long-term memories in the brain.

Amino Acid Sequence↗

PubMed2003

A global representation of the protein fold space.

One of the principal goals of the structural genomics initiative is to identify the total repertoire of protein folds and obtain a global view of the "protein structure universe." Here, we present a 3D map of the protein fold space in which structurally related folds are represented by spatially adjacent points. Such a representation reveals a high-level organization of the fold space that is intuitively interpretable. The shape of the fold space and the overall distribution of the folds are defined by three dominant trends: secondary structure class, chain topology, and protein domain size. Random coil-like structures of small proteins and peptides are mapped to a region where the three trends converge, offering an interesting perspective on both the demography of fold space and the evolution of protein structures.

Bacterial Proteins↗

PubMed2003

A survey of arsenic species in Chinese seafood.

In the present report, thirty different types of Chinese edible seafood, including brown algae, red algae, fish, crab, shrimp, mussels, oysters, and clams, which are very popular foodstuffs in the Chinese kitchen, were examined for their total content of As as well as its different species. Total arsenic concentration in algae samples was 1.7-38.7 microg/g (dry weight), and 0.086-7.54 microg/g in fish and shellfish (wet weight), respectively. The arsenic species in seafood extracts were determined by using anion and cation exchange high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS). Arsenosugars were detected in all of the extracted algae samples (1.5-33.8 microg/g dry weight) and fish samples (0.018-0.78 microg/g wet weight). Arsenobetaine was detected in all of the extracted fish and shellfish samples (0.025-6.604 microg/g wet weight). In contrast, inorganic arsenic in fish and shellfish samples occurred at levels below 2% of the total arsenic. No inorganic arsenic was detected in the algae samples. This study provides information about the distribution pattern of arsenic species in seafood products. Since the major share of arsenic components in seafood is organic arsenic with a low toxicity, we can conclude that arsenic in seafood does not pose any risk to human health.

Arsenicals↗

PubMed2003

Overview of structural genomics: from structure to function.

The unprecedented increase in the number of new protein sequences arising from genomics and proteomics highlights directly the need for methods to rapidly and reliably determine the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds, thereby providing three-dimensional portraits for all proteins in a living organism and to infer molecular functions of the proteins. The goal of obtaining protein structures on a genomic scale has motivated the development of high-throughput technologies for macromolecular structure determination, which have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional and evolution relationships that were hidden at the sequence level.

Amino Acid Sequence↗

PubMed2003

Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy.

Macroautophagy is a catabolic membrane trafficking phenomenon that is observed in all eukaryotic cells in response to various stimuli, such as nitrogen starvation and challenge with specific hormones. In the yeast Saccharomyces cerevisiae, the induction of autophagy involves a direct signal transduction mechanism that affects membrane dynamics. In this system, the induction process modifies a constitutive trafficking pathway called the cytoplasm-to-vacuole targeting (Cvt) pathway, which transports the vacuolar hydrolase aminopeptidase I, from the formation of small Cvt vesicles to the formation of autophagosomes. Apg1 is one of the proteins required for the direct signal transduction cascade that modifies membrane dynamics. Although Apg1 is required for both the Cvt pathway and autophagy, we find that Apg1 kinase activity is required only for Cvt trafficking of aminopeptidase I but not for import via autophagy. In addition, the data support a novel role for Apg1 in nucleation of autophagosomes that is distinct from its catalytic kinase activity and imply a qualitative difference in the mechanism of autophagosome and Cvt vesicle formation.

Aminopeptidases↗

PubMed2003

Gene therapy for gastric cancer: a review.

Gastric cancer is common in China, and its early diagnosis and treatment are difficult. In recent years great progress has been achieved in gene therapy, and a wide array of gene therapy systems for gastric cancer has been investigated. The present article deals with the general principles of gene therapy and then focuses on how these principles may be applied to gastric cancer.

Genetic Therapy↗

PubMed2003

Repair of radius defect with bone-morphogenetic-protein loaded hydroxyapatite/collagen-poly(L-lactic acid) composite.

OBJECTIVE: To explore the method to repair bone defect with bone-morphogenetic-protein loaded hydroxyapatite/collagen-poly(L-lactic acid) composite. METHODS: 18 adult beagle dogs were randomly divided into 3 groups. In Group A, bone-morphogenetic-protein (BMP) loaded hydroxyapatite/collagen-poly(L-lactic acid) (HAC-PLA) scaffold was implanted in a 2 cm diaphyseal defect in the radius. In Group B, unloaded pure HAC-PLA scaffold was implanted in the defects. No material was implanted in Group C (control group). The dogs were sacrificed 6 months postoperatively. Features of biocompatibility, biodegradability and osteoinduction were evaluated with histological, radiological examinations and bone mineral density (BMD) measurements. RESULTS: In Group A, the radius defect healed after the treatment with BMP loaded HAC-PLA. BMD at the site of the defect was higher than that of the contralateral radius. Fibrous union developed in the animals of the control group. CONCLUSIONS: BMP not only promotes osteogenesis but also accelerates degradation of the biomaterials. Optimized design parameters of a three-dimensional porous biomaterial would give full scope to the role of BMP as an osteoinductive growth factor.

Animals↗

PubMed2003

[The postburn changes in gastric mucosal blood flow and Na+-K+-ATPase activity and their effects on gastric transmucosal potential difference in severely scalded rats].

OBJECTIVE: To investigate the postburn changes in gastric mucosal blood flow (GMBF) and Na(+)-K(+)-ATPase activity and their effects on gastric transmucosal potential difference (GTPD) in severely scalded rats. METHODS: Rats were inflicted with hot water scald on the back. Laser Doppler blood flow meter, electric physiological recorder meter and biochemical method were employed in the study to determine the changes in GMBF, GTPD and gastric mucosal Na(+)-K(+)-ATPase activity in the rats before injury and at 3, 6, 12, 24 and 48 PBHs. Normal rats served as controls, The above indices were also detected at the corresponding time points. All the data was collected and analyzed for their correlation. RESULTS: The GMBF and Na(+)-K(+)-ATPase activity was at 3 - 24 PBHs were evidently lower in the scalded rats than those in controls (P < 0.05 - 0.01). GTPD was decreased significantly at 6 - 48 PBHs (P < 0.05 - 0.01). was above three indices reached the lowest values at 12 PBH. By correlation analysis the results indicated that the Na(+)-K(+)-ATPase activity The decreased in accordance with a decrease in GMBF (r = 0.527, P < 0.01). The GTPD decrease could both be induced by GMBF decrease and Na(+)-K(+)-ATPase activity decrease (r = 0.453 and 0.527, respectively, P < 0.01). CONCLUSION: The decrease in GMBF and Na(+)-K(+)-ATPase activity occurred in severely scalded rats, both changes might be the major cause of postburn gastric mucosal barrier injury.

Animals↗

PubMed2003

[Chi-squared automatic interaction detection analysis and its application in evaluating factors attributing to Chinese university teachers' professional burnout].

OBJECTIVE: To introduce Chi-squared automatic interaction detection (CHAID) method and its application in assessment of the factors that contribute to the Chinese university teachers' professional burnout. METHOD: With CHAID method, the data acquired from a questionnaire-based investigation among 100 full-time teachers of Chinese universities were analyzed. RESULTS AND CONCLUSION: Significant difference in the attitude toward the profession was noted between the teachers with high- and low-level professional burnout. Among the factors contributing to the teachers' negative attitude, the sense of accomplishment in the profession, degree of preference of the profession, status of personal relations and social communications, along with the working environment, are the key elements that have most profound effect upon the teachers.

Burnout, Professional↗

PubMed2003

[Studies on the chemical constituents of Chinese herb Melastoma dodecandrum].

OBJECTIVE: To investigate the chemical constituents of Chinese herb Melastoma dodecandrum. METHOD: The chemical constituents were isolated by solvent extraction and chromatography. Their structure were identified on the basis of physic-chemical constants and specral data. RESULT: Five compounds were isolated and identified as hexacosanoic acid (I), quercetin(II), avicularin(III), gallic acid (IV), kaempferol(V). CONCLUSION: The above-mentioned compounds were separated from the plant for the first time.

Fatty Acids↗

PubMed2002

A new europium beta-diketone chelate for ultrasensitive time-resolved fluorescence immunoassays.

4,4'-Bis(1",1",1"-trifluoro-2",4"-butanedione-6"-yl)-chlorosulfo-o-terphenyl (BTBCT) was synthesized by modifying the structure of the reported BHHCT. In comparison with the original BHHCT, the detection sensitivity of BTBCT-Eu chelate in aqueous solution was improved approximately 8 times by time-resolved fluorescence measurement. To construct sensitive TRFIAs with the use of BTBCT-Eu chelate as the fluorescent label, streptavidin-BSA conjugate was prepared by the maleimide-thiol method and labeled by BTBCT. The streptavidin-BSA conjugate and its BTBCT-labeled complex were affinity-purified using 2-iminobiotin-agarose as binding reagent. With streptavidin-BSA-BTBCT-Eu complex as signal generation reagent, a highly sensitive indirect serum hTSH TR-IFMA was developed. The low limit of detection (LLD) of the TSH TR-IFMA was 0.011 mIU/L with 10 microl of sample volume, corresponding to approximately 337,900 molecules per test. To evaluate the utility of BTBCT-Eu label in direct TRFIAs, a competitive serum T4 TRFIA was developed with T4-BSA-BTBCT-Eu complex as competing tracer. The measurements obtained by the present TSH TR-IFMA or T4 TRFIA correlated well with those obtained by commercial Wallac TSH DELFIA Ultra or T4 DELFIA, respectively. Primary results show that BTBCT can be employed as a powerful labeling material for constructing ultrasensitive TRFIAs.

Animals↗

PubMed2002

Synthesis of a highly fluorescent beta-diketone-europium chelate and its utility in time-resolved fluoroimmunoassay of serum total thyroxine.

A new highly fluorescent beta-diketone-europium chelate was synthesized and employed as a tracer to develop a time-resolved fluoroimmunoassay (TRFIA) for detection of serum total thyroxine (T4). The tetradentate beta-diketone chelator, 1,10-bis(thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BTOT), was structurally composed of two units of thenoyltrifluoroacetone (TTA) derivatives but expressed fluorescence that was greatly enhanced, as compared to the original TTA molecules, in the presence of excess amount of Eu3+. The luminescence properties of the europium chelate of BTOT werestudied in aqueous solution. Chlorosulfonylation of BTOT afforded 1, 10-bis(5'-chlorosulfo-thiophene-2'-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BCTOT), which could be coupled to proteins (i.e., streptavidin or the BSA-T4 conjugate) and used as a tracer for TRFIA. Although the BCTOT-Eu complex could be detected at a very low level (approximately 1.07 x 10(-12) mol/L) in buffered aqueous solution (50 mmoVLTris-HCl; pH, 8.0), the application of the chelate label in direct serum T4 TRFIA experienced a problem of matrix interference, which was probably caused by some unknown chelating components in the samples as a result of the fact that the fluorescence of the BCTOT-Eu chelate was prone to quenching or enhancement by some chelating reagents. To remove this problem, an indirect serum T4 TRFIA was proposed with the use of BCTOT-Eu-labeled streptavidin (SA) as signal generation reagent. The concentrations of T4 in 27 human serums were determined by indirect T4 TRFIA, and the assay results correlated well with those obtained by commercial Coming-CLIA (r = 0.955) and Wallac-DELFIA (r 0.965).

Chelating Agents↗

PubMed2002

Determination of ethamsylate in pharmaceutical preparations based on an auto-oxidation chemiluminescence reaction.

Strong chemiluminescence emission has been observed by mixing alkaline hydrolytic products of ethamsylate with Tween 80 in acidic rhodamine 6G solution. This phenomenon has been utilized to design a flow-injection chemiluminescence method for the determination of ethamsylate in a pharmaceutical preparation. Under the optimum conditions, the proposed procedure has a linear range between 0.05 and 2.0 microg ml(-1), with a detection limit of 0.02 microg ml(-1) for ethamsylate. The method was applied to the determination of ethamsylate in pharmaceutical preparations. The possible mechanism of this chemiluminescence reaction was proposed.

Ethamsylate↗

PubMed2002

The Saccharomyces cerevisiae Mob2p-Cbk1p kinase complex promotes polarized growth and acts with the mitotic exit network to facilitate daughter cell-specific localization of Ace2p transcription factor.

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. We investigated the function of two S. cerevisiae proteins related to the MEN proteins Mob1p and Dbf2p kinase. Previous work indicates that cells lacking the Dbf2p-related protein Cbk1p fail to sustain polarized growth during early bud morphogenesis and mating projection formation (Bidlingmaier, S., E.L. Weiss, C. Seidel, D.G. Drubin, and M. Snyder. 2001. Mol. Cell. Biol. 21:2449-2462). Cbk1p is also required for Ace2p-dependent transcription of genes involved in mother/daughter separation after cytokinesis. Here we show that the Mob1p-related protein Mob2p physically associates with Cbk1p kinase throughout the cell cycle and is required for full Cbk1p kinase activity, which is periodically activated during polarized growth and mitosis. Both Mob2p and Cbk1p localize interdependently to the bud cortex during polarized growth and to the bud neck and daughter cell nucleus during late mitosis. We found that Ace2p is restricted to daughter cell nuclei via a novel mechanism requiring Mob2p, Cbk1p, and a functional nuclear export pathway. Furthermore, nuclear localization of Mob2p and Ace2p does not occur in mob1-77 or cdc14-1 mutants, which are defective in MEN signaling, even when cell cycle arrest is bypassed. Collectively, these data indicate that Mob2p-Cbk1p functions to (a) maintain polarized cell growth, (b) prevent the nuclear export of Ace2p from the daughter cell nucleus after mitotic exit, and (c) coordinate Ace2p-dependent transcription with MEN activation. These findings may implicate related proteins in linking the regulation of cell morphology and cell cycle transitions with cell fate determination and development.

Active Transport, Cell Nucleus↗

PubMed2002

Chemical genetic blockade of transformation reveals dependence on aberrant oncogenic signaling.

BACKGROUND: Our understanding of protein kinase inhibition in the treatment of cancer is clearly limited by the lack of inhibitors that selectively block a single kinase implicated in neoplastic transformation. One approach to developing specific inhibitors is to engineer in protein kinases silent mutations that allow selective inhibition while retaining kinase activity. Because it is implicated in a large number of malignancies, EGFR provides an attractive target for such selective kinase inhibition. RESULTS: We generated an inhibitor-sensitized allele of the transforming receptor tyrosine kinase v-erbB. Transformation of immortalized rodent fibroblasts by sensitized versions of v-erbB (v-erbB-as1) was blocked by 1-napthyl PP1 (NaPP1), a cell-permeable ATP-competitive inhibitor. NaPP1 also reversed morphological transformation by v-erbB-as1. Signaling through MAP kinase and PI(3) kinase was initially blocked by inhibitor treatment and then recovered to levels comparable to those in nontransformed cells. Surprisingly, NaPP1-treated v-erbB-as1 cells failed to re-enter the cell cycle, showed decreased levels of D- and A-type cyclins, and showed increased levels of p27. To extend this result, we showed that NaPP1 treatment of v-Src-as1 cells also led to cell cycle arrest. Arrested cells could be rescued with a conditional allele of Raf or by transduction of a constitutive allele of cyclin D1. CONCLUSIONS: These data suggest that mammalian cells can become dependent on aberrant oncogenic signaling; this dependency renders them incapable of returning to a normal, proliferative phenotype.

3T3 Cells↗

PubMed2002

Protein engineering of protein kinase A catalytic subunits results in the acquisition of novel inhibitor sensitivity.

Analysis of the role of specific protein kinases in signal transduction networks has relied heavily on ATP analog inhibitors. Currently used agents, however, often do not distinguish between kinase family members. Genetic approaches can also be used to inactivate a specific kinase, but these techniques do not afford the rapid kinetics possible with pharmacological inhibitors. To circumvent this problem, modification of the structure of a particular protein kinase can be performed to engineer a drug-target interaction of choice. We have used this method to create protein kinase A (PKA) catalytic subunits with modifications that confer sensitivity to novel ATP analog inhibitors. Mutation of methionine 120 to alanine or glycine in either the Calpha or Cbeta subunits of PKA induces sensitivity to a series of C-3 derivatized pyrazolo[3,4-d]pyrimidine-based inhibitors. Modification of threonine 183 enhances this inhibitor sensitivity. The IC(50) values in cell culture of the most broadly effective agent, 1-NM, ranged from 25 to 200 nm depending upon the combination of modified amino acids and were significantly higher than the potencies observed with H-89. Despite their high sequence conservation, Cbeta enzymes with inhibitor-sensitive amino acids at position 120 showed a substantial loss of overall catalytic activity when used to induce reporter gene transcription in transfected cells. Conversion of position 46 (lysine to isoleucine) rescued the ability of position 120 mutated Cbeta enzymes to induce gene transcription. Application of this combined genetic and pharmacological approach should allow analysis of the specific roles of PKA isoforms in cell culture and in vivo.

Alanine↗