Biomedical subjects

H Ma

Publications and source records attributed to H Ma.

At least 127 records · Page 7Linked to original sources

PubMed2000

[Sequential injection vapor generation atomic absorption spectrometric determination of mercury and arsenic].

A sequential injection vapor generation atomic absorption spectrometric (SI-VGAAS) method for the determination of mercury and arsenic was developed, characterized by reagent merging using a double-syringe pump and a multi-position selector valve. A sample throughput of 120 h-1 was achieved with 500 microL sample with a precision of 2.2% for mercury at 8.0 micrograms.L-1 (n = 11) and 1.4% for arsenic at 6.0 micrograms.L-1 (n = 11) levels and a detection limit of 0.15 microgram.L-1 for mercury and 0.07 microgram.L-1 for arsenic. The method was applied to the analysis of geochemical standard reference material GSS-5, and good agreement with the certified values were obtained.

Arsenic↗

PubMed2000

[Conformity of cancerous diagnosis of serum-fluorimetry under the influence of carotenoid fluorescence].

We observed that carotenoid had sensitization for fluorescence of porphyrin as cancerous diagnostic technology was applied in clinic by serum-fluorimetry. The Forster theory was used in order to analyse sensitized fluorescence between carotenoid and porphyrin in this paper. Transfer characteristic between energy donor and energy acceptor was calculated and discussed its influence on cancerous diagnosis.

Biomarkers, Tumor↗

PubMed1999

Overlapping effects of S3 stalk segment mutations on the affinity of Ca2+-ATPase (SERCA) for thapsigargin and cyclopiazonic acid.

Chimeric exchanges and mutations were produced in the Ca(2+)-ATPase (SERCA) to match (in the majority of cases) corresponding sequences of the Na(+),K(+)-ATPase. The effects of these mutations on the concentration dependence of the specific Ca(2+)-ATPase inhibition by thapsigargin (TG) and cyclopiazonic acid (CPA) were then determined. Extensive chimeric mutations on the large cytosolic loop, on the S4 stalk segment, and on the M3 transmembrane segments produced little or no modification of the Ca(2+)-ATPase sensitivity to either inhibitor. On the other hand, the presence of a six amino acid Na(+), K(+)-ATPase sequence within the S3 stalk segment of the Ca(2+)-ATPase raised 60-fold the apparent K(i) for TG and 250-fold the apparent K(i) for CPA. More limited mutations within the same S3 segment, however, affected differently the concentration dependence of the Ca(2+)-ATPase inhibition by TG or CPA. Specifically, single mutation of Phe256 to Val increased 20-fold the apparent K(i) for TG, while having very little effect on the apparent K(i) for CPA. These findings indicate significant overlap of the TG and CPA binding domains within the S3 stalk segment of the Ca(2+)-ATPase, where the contribution of each protein residue is dependent on the structures of the two inhibitors. Saturating concentrations of either or both TG and CPA produce an identical reduction of the affinity of the ATPase for ATP, suggesting that only one inhibitor can bind at any time due to significant overlap of their binding domains. It is suggested that perturbations produced by binding of either inhibitor within the stalk segment interfere with the long-range functional linkage between ATP utilization in the ATPase cytosolic region and Ca(2+) binding in the membrane-bound region.

Adenosine Triphosphate↗

PubMed1999

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: diverse roles for mitogen-activated protein kinase homologs in foliar pathogens.

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Deltachk1 mutants is attempted. The ability of Deltachk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

Amino Acid Sequence↗

PubMed1999

Conversion of mammalian 3alpha-hydroxysteroid dehydrogenase to 20alpha-hydroxysteroid dehydrogenase using loop chimeras: changing specificity from androgens to progestins.

Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3alpha-HSD catalyzes the inactivation of androgens in the prostate by converting 5alpha-dihydrotestosterone to 3alpha-androstanediol, where excess 5alpha-dihydrotestosterone is implicated in prostate disease. By contrast, 20alpha-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20alpha-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3alpha-HSDs and 20alpha-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3alpha-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3alpha-HSD was converted to 20alpha-HSD by replacing these loops with those found in 20alpha-HSD. However, when pocket residues in 3alpha-HSD were mutated to those found in 20alpha-HSD altered specificity was not achieved. Replacement of loop A created a 17beta-HSD activity that was absent in either 3alpha- or 20alpha-HSD. Once loops A and C were replaced, the chimera had both 3alpha- and 20alpha-HSD activity. When loops A, B, and C were substituted, 3alpha-HSD was converted to a stereospecific 20alpha-HSD with a resultant shift in k(cat)/K(m) for the desired reaction of 2 x 10(11). This study represents an example where sex hormone specificity can be changed at the enzyme level.

20-Hydroxysteroid Dehydrogenases↗

PubMed1999

The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation.

The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine Arabidopsis SKP1 homologues have been uncovered and are named ASK1 through ASK9. Here, we report the isolation and characterization of a male sterile Arabidopsis mutant and show that the mutant defect was caused by a Ds transposon insertion into the ASK1 gene. In the ask1-1 mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appeared normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in ask1-1, at least some homologues remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, we propose that ASK1 controls homologue separation by degrading or otherwise removing a protein that is required directly or indirectly for homologue association before anaphase I.

Amino Acid Sequence↗

PubMed1999

Seed development: with or without sex?

In Arabidopsis, mutations in the genes FIE, FIS2 or MEDEA disrupt the normal dependence of seed development on fertilization. The products of these genes are similar to Polycomb group proteins, suggesting possible mechanisms for determining whether seeds can be produced sexually or asexually.

Arabidopsis↗

PubMed1999

Association of chromosome territories with the nuclear matrix. Disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteins.

To study the possible role of the nuclear matrix in chromosome territory organization, normal human fibroblast cells are treated in situ via classic isolation procedures for nuclear matrix in the absence of nuclease (e.g., DNase I) digestion, followed by chromosome painting. We report for the first time that chromosome territories are maintained intact on the nuclear matrix. In contrast, complete extraction of the internal nuclear matrix components with RNase treatment followed by 2 M NaCl results in the disruption of higher order chromosome territory architecture. Correlative with territorial disruption is the formation of a faint DNA halo surrounding the nuclear lamina and a dispersive effect on the characteristically discrete DNA replication sites in the nuclear interior. Identical results were obtained using eight different human chromosome paints. Based on these findings, we developed a fractionation strategy to release the bulk of nuclear matrix proteins under conditions where the chromosome territories are maintained intact. A second treatment results in disruption of the chromosome territories in conjunction with the release of a small subset of acidic proteins. These proteins are distinct from the major nuclear matrix proteins and may be involved in mediating chromosome territory organization.

Ammonium Sulfate↗

PubMed1999

Characterization of homogeneous recombinant rat ovarian 20alpha-hydroxysteroid dehydrogenase: fluorescent properties and inhibition profile.

In rat ovary, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, converts progesterone into the inactive progestin 20alpha-hydroxyprogesterone and has been implicated in the termination of pregnancy. Here we report a convenient overexpression system that permits the purification of milligram quantities of homogeneous recombinant 20alpha-HSD with wild-type enzyme activity. The availability of this enzyme has permitted detailed kinetic, inhibition and fluorescence analyses. The enzyme exhibited narrow steroid specificity, catalysing reactions only at C-20; it reduced progesterone and 17alpha-hydroxyprogesterone and oxidized 20alpha-hydroxypregnanes. It also turned over common AKR substrates, such as 9, 10-phenanthrenequinone and 4-nitrobenzaldehyde. The intrinsic fluorescence spectrum of 20alpha-HSD was characterized and was quenched on the binding of NADP(H), yielding a KNADPd of 0.36 microM and a KNADPHd of 0.64 microM. NADP(H) binding generated an energy transfer band that could not be quenched by steroids. Inhibition studies conducted with non-steroidal and steroidal anti-inflammatory drugs and synthetic oestrogens indicated that even though rat ovarian 20alpha-HSD and rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) share more than 67% amino acid identity, their inhibition profiles are markedly different. Unlike 3alpha-HSD, most of these compounds did not inhibit 20alpha-HSD. Only meclofenamic acid and hexoestrol were potent competitive inhibitors for 20alpha-HSD, yielding K(i) values of 18.9 and 14.3 microM respectively. These studies suggest that selective non-steroidal AKR inhibitors could be developed for 20alpha-HSD that might be useful in maintaining pregnancy and that specific inhibitors might be developed from either N-phenylanthranilates or biphenols.

20-Hydroxysteroid Dehydrogenases↗

PubMed1999

Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana.

The plant hormone auxin regulates diverse aspects of plant growth and development. We report that in Arabidopsis, auxin response is dependent on a ubiquitin-ligase (E3) complex called SCFTIR1. The complex consists of proteins related to yeast Skp1p and Cdc53p called ASK and AtCUL1, respectively, as well as the F-box protein TIR1. Mutations in either ASK1 or TIR1 result in decreased auxin response. Further, overexpression of TIR1 promotes auxin response suggesting that SCFTIR1 is limiting for the response. These results provide new support for a model in which auxin action depends on the regulated proteolysis of repressor proteins.

Amino Acid Sequence↗

PubMed1999

Regulation of transcription by a protein methyltransferase.

The p160 family of coactivators, SRC-1, GRIP1/TIF2, and p/CIP, mediate transcriptional activation by nuclear hormone receptors. Coactivator-associated arginine methyltransferase 1 (CARM1), a previously unidentified protein that binds to the carboxyl-terminal region of p160 coactivators, enhanced transcriptional activation by nuclear receptors, but only when GRIP1 or SRC-1a was coexpressed. Thus, CARM1 functions as a secondary coactivator through its association with p160 coactivators. CARM1 can methylate histone H3 in vitro, and a mutation in the putative S-adenosylmethionine binding domain of CARM1 substantially reduced both methyltransferase and coactivator activities. Thus, coactivator-mediated methylation of proteins in the transcription machinery may contribute to transcriptional regulation.

Amino Acid Sequence↗

PubMed1999

The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3 alpha-hydroxysteroid dehydrogenase, a representative aldo-keto reductase.

Fluorescence stopped-flow studies were conducted with recombinant rat liver 3 alpha-HSD, an aldo-keto reductase (AKR) that plays critical roles in steroid hormone inactivation, to characterize the binding of nicotinamide cofactor, the first step in the kinetic mechanism. Binding of NADP(H) involved two events: the fast formation of a loose complex (E.NADP(H)), followed by a conformational change in enzyme structure leading to a tightly bound complex (E.NADP(H)), which was observed as a fluorescence kinetic transient. Binding of NAD(H) was not characterized by a similar kinetic transient, implying a difference in the mode of binding of the two cofactors. Unlike previously characterized AKRs, the rates associated with the formation and decay of E.NADP(H) and E.NADP(H) were much faster than kcat for the oxidoreduction of various substrates, indicating that binding and release of cofactor is not rate-limiting overall in 3 alpha-HSD. Mutation of Arg 276, a highly conserved residue in AKRs that forms a salt bridge with the adenosine 2'-phosphate of NADP(H), resulted in large changes in Km and Kd for NADP(H) that were not observed with NAD(H). The loss in free energy associated with the increase in Kd for NADP(H) is consistent with the elimination of an electrostatic link. Importantly, this mutation abolished the kinetic transient associated with NADPH binding. Thus, anchoring of the adenosine 2'-phosphate of NADPH by Arg 276 appears to be obligatory for the fluorescence kinetic transients to be observed. The removal of Trp 86, a residue involved in fluorescence energy transfer with NAD(P)H, also abolished the kinetic transient, but mutation of Trp 227, a residue on a mobile loop associated with cofactor binding, did not. It is concluded that in 3 alpha-HSD, the time dependence of the change in Trp 86 fluorescence is due to cofactor anchoring, and thus, Trp 86 is a distal reporter of this event. Further, the loop movement that accompanies cofactor binding is spectrally silent.

3-Hydroxysteroid Dehydrogenases↗

PubMed1999

Regulation of Dictyostelium protein-tyrosine phosphatase-3 (PTP3) through osmotic shock and stress stimulation and identification of pp130 as a PTP3 substrate.

Osmotic shock and growth-medium stimulation of Dictyostelium cells results in rapid cell rounding, a reduction in cell volume, and a rearrangement of the cytoskeleton that leads to resistance to osmotic shock. Osmotic shock induces the activation of guanylyl cyclase, a rise in cGMP mediating the phosphorylation of myosin II, and the tyrosine phosphorylation of actin and the approximately 130-kDa protein (p130). We present data suggesting that signaling pathways leading to these different responses are, at least in part, independent. We show that a variety of stresses induce the Ser/Thr phosphorylation of the protein-tyrosine phosphatase-3 (PTP3). This modification does not alter PTP3 catalytic activity but correlates with its translocation from the cytosol to subcellular structures that co-localize to endosomal vesicles. This translocation is independent of PTP3 activity. Mutation of the catalytically essential Cys to a Ser results in inactive PTP3 that forms a stable complex with tyrosine-phosphorylated p130 (pp130) in vivo and in vitro, suggesting that PTP3 has a substrate specificity for pp130. The data suggest that stresses activate several interacting signaling pathways controlled by Ser/Thr and Tyr phosphorylation, which, along with the activation of guanylyl cyclase, mediate the ability of this organism to respond to adverse changes in the external environment.

Actins↗

PubMed1999

Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.

Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein-coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation-stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.

Amino Acid Sequence↗

PubMed1999

[Study of NAIP gene in spinal muscular atrophy]

OBJECTIVE: Investigating the correlation between the genotype and spinal muscular atrophy(SMA) clinical phenotype. METHODS: Neuronal apoptosis inhibitory protein(NAIP) gene analysis was performed by PCR amplification of exon 5 in 13 unrelated SMA patients with deletions of SMN gene. RESULTS: Two Type-I patients(2/5, 40%) lacked the NAIP exon 5. CONCLUSION: The results suggest a possible correlation between NAIP gene deletion and the disease severity of SMA.

Journal Article↗

PubMed1999

[Mutations of the fibroblast growth factor receptor 3 gene in achondroplasia].

OBJECTIVE: To evaluate whether mutation in the exon 10 of the fibroblast growth factor receptor 3(FGFR3) gene in common in Chinese patients with achondroplasia. METHODS: Genomic DNA from seven sporadic cases of achondroplasia was studied by using PCR-SSCP and restriction enzymes. RESULTS: All patients carried the same glycine-to-arginine mutation at codon 380(G380R) of the transmembrane domain of FGFR3. CONCLUSION: The results suggest that the G380R mutation of FGFR3 is also a frequent mutation causing achondroplasia in Chinese. Detecting FGFR 3 gene mutation with PCR-SSCP and restriction enzymes analysis is a convenient, rapid and reliable molecular diagnostic assay for prenatal and early diagnosis of achondroplasia.

Achondroplasia↗

PubMed1999

An additional region of coactivator GRIP1 required for interaction with the hormone-binding domains of a subset of nuclear receptors.

Transcriptional coactivators of the p160 family (SRC-1, GRIP1, and p/CIP) associate with DNA-bound nuclear receptors (NRs) and help the NRs to recruit an active transcription initiation complex to the promoters of target genes. Previous studies have demonstrated the importance of the NR interaction domain (NID) of p160 proteins containing three NR box motifs (LXXLL) for the interaction with the hormone-binding domains of NRs. Here we report that, in addition to NID, another region of coactivator GRIP1 (amino acids 1011-1121), called the auxiliary NID (NIDaux), is required in vitro and in vivo for efficient interaction with a subset of NRs, including the glucocorticoid receptor (GR), androgen receptor, and retinoic acid receptor alpha. A second group of NRs, which includes the progesterone receptor, retinoid X receptor alpha, thyroid hormone receptor beta1, and vitamin D receptor, required only NID for efficient interaction. For binding to GR, the NID and NIDaux of GRIP1 must act in cis, but deletion of up to 144 amino acids between the two regions did not reduce binding efficiency. Amino acids 1011-1121 of GRIP1 also contain a p300 interaction domain, but mutational analysis indicated that the p300 interaction function within this region is separable from the ability to contribute to GR hormone-binding domain binding. SRC-1 lacks an NIDaux activity equivalent to that in GRIP1.

Amino Acid Sequence↗

PubMed1999

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo.

Arabidopsis↗