Biomedical subjects

R Mei

Publications and source records attributed to R Mei.

11 recordsLinked to original sources

PubMed2000

RNA expression analysis using a 30 base pair resolution Escherichia coli genome array.

We have developed a high-resolution "genome array" for the study of gene expression and regulation in Escherichia coli. This array contains on average one 25-mer oligonucleotide probe per 30 base pairs over the entire genome, with one every 6 bases for the intergenic regions and every 60 bases for the 4,290 open reading frames (ORFs). Twofold concentration differences can be detected at levels as low as 0.2 messenger RNA (mRNA) copies per cell, and differences can be seen over a dynamic range of three orders of magnitude. In rich medium we detected transcripts for 97% and 87% of the ORFs in stationary and log phases, respectively. We found that 1, 529 transcripts were differentially expressed under these conditions. As expected, genes involved in translation were expressed at higher levels in log phase, whereas many genes known to be involved in the starvation response were expressed at higher levels in stationary phase. Many previously unrecognized growth phase-regulated genes were identified, such as a putative receptor (b0836) and a 30S ribosomal protein subunit (S22), both of which are highly upregulated in stationary phase. Transcription of between 3,000 and 4,000 predicted ORFs was observed from the antisense strand, indicating that most of the genome is transcribed at a detectable level. Examples are also presented for high-resolution array analysis of transcript start and stop sites and RNA secondary structure.

Escherichia coli↗

PubMed2000

Genome-wide detection of allelic imbalance using human SNPs and high-density DNA arrays.

Most human cancers are characterized by genomic instability, the accumulation of multiple genetic alterations and allelic imbalance throughout the genome. Loss of heterozygosity (LOH) is a common form of allelic imbalance and the detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes and to characterize tumor stages and progression. Here we describe the use of high-density oligonucleotide arrays for genome-wide scans for LOH and allelic imbalance in human tumors. The arrays contain redundant sets of probes for 600 genetic loci that are distributed across all human chromosomes. The arrays were used to detect allelic imbalance in two types of human tumors, and a subset of the results was confirmed using conventional gel-based methods. We also tested the ability to study heterogeneous cell populations and found that allelic imbalance can be detected in the presence of a substantial background of normal cells. The detection of LOH and other chromosomal changes using large numbers of single nucleotide polymorphism (SNP) markers should enable identification of patterns of allelic imbalance with potential prognostic and diagnostic utility.

Adenocarcinoma↗

PubMed1996

Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing.

Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.

Animals↗

PubMed1996

[Construction of shuttle vector containing delta-endotoxin gene of Bacillus thuringiensis].

The Bacillus thuringiensis toxin gene CryIA(c) was inserted into the shuttle vector pBE-2 to construct pAMY for expressing the B.t. gene in both Gram-negative and -positive bacterial systems. pAMY was introduced into wild type Bacillus cereus, B.brevis and B.subtilis by electroporation. Transformants containing delta-endotoxin gene produced proteins reacted with B.t. crystal protein antibody. Upon biological toxicity tests, the transformants gave a mortality of 100% against Ostrinia furnacilis, 58.8% against Heliothis armigera and 100% against Heliothis assulta. The ability of promoting plant growth of the original strains is retained.

Bacillus cereus↗

PubMed1994

Transfer of shuttle vectors containing Bacillus thuringiensis toxin gene into wild-type B. cereus, B. brevis and B. subtilis by electroporation.

Gram positive and negative bacterial shuttle vectors carrying Bacillus thuringiensis (B.t.) toxin gene were introduced by electroporation into wild-type Bacillus cereus, B. brevis and B. subtilis. The transformation efficiencies for these bacteria were around 10(1)-10(4) transformants per micrograms DNA based on the numbers of neomycin- and ampicillin-resistant colonies produced. The structure of the transferred plasmids proved identical with the original ones both in size and restriction pattern. Toxicity assays showed that the transformants gave a mortality of 90-100% against caterpillar of Heliothis assulta, indicating that the gene function was not changed by electroporation.

Bacillus↗

PubMed1992

Comparative properties of hydroquinone and hydroxylamine reduction of the Ca(2+)-stabilized O2-evolving complex of photosystem II: reductant-dependent Mn2+ formation and activity inhibition.

Calcium binding to photosystem II slows NH2OH inhibition of O2 evolution; Mn2+ is retained by the O2-evolving complex [Mei, R., & Yocum, C. F. (1991) Biochemistry 30, 7836-7842]. This Ca(2+)-induced stability has been further characterized using the large reductant hydroquinone. Salt-washed photosystem II membranes reduced by hydroquinone in the presence of Ca2+ retain 80% of steady-state O2 evolution activity and contain about 2 Mn2+/reaction center that can be detected at room temperature by electron paramagnetic resonance. This Mn2+ produces a weak enhancement of H2O proton spin-lattice relaxation rates, cannot be easily extracted by a chelator, and is reincorporated into the O2-evolving complex upon illumination. A comparison of the properties of Ca(2+)-supplemented photosystem II samples reduced by hydroquinone or NH2OH alone or in sequence reveals the presence of a subpopulation of manganese atoms at the active site of H2O oxidation that is not accessible to facile hydroquinone reduction. At least one of these manganese atoms can be readily reduced by NH2OH following a noninhibitory hydroquinone reduction step. Under these conditions, about 3 Mn2+/reaction center are lost and O2 evolution activity is irreversibly inhibited. We interpret the existence of distinct sites of reductant action on manganese as further evidence that the Ca(2+)-binding site in photosystem II participates in regulation of the organization of manganese-binding ligands and the overall structure of the O2-evolving complex.

Binding Sites↗

PubMed1992

Chinese medical school exit objectives: a multi-institutional survey of teacher opinion.

This paper describes a collaborative survey of Chinese medical school teacher opinion concerning clinical behaviours and skills that Chinese students should be expected to demonstrate prior to graduation from medical school. Selected teachers from four Chinese medical schools indicated whether each of 77 objectives was: essential for every student to know or demonstrate prior to graduation; useful but not essential; or not applicable to their educational programmes. Results show teachers' expectations regarding some of the behaviours and skills Chinese medical school teachers consider to be important for students to learn. Forty-nine per cent of the exit objectives were regarded as essential by 75% or more of the teachers. The most essential objectives involved performing clinical procedures, collaborating and communicating with health care professionals, conducting organ system examinations, formulating hypotheses and defining problems. Other essential objectives were concerned with communicating with patients and their families, maintaining currency within the discipline, and recognition, evaluation and triage of patients with life-threatening or chronic conditions. Exit objectives related to health promotion and risk assessment were regarded as useful but not essential at the undergraduate level. Implications for Chinese medical education are discussed.

China↗

PubMed1991

Calcium retards NH2OH inhibition of O2 evolution activity by stabilization of Mn2+ binding to photosystem II.

Calcium is required for oxidation of water to molecular oxygen by photosystem II; the Ca2+ demand of the reaction increases upon removal of 23- and 17-kDa extrinsic polypeptides from detergent-derived preparations of the photosystem. Employing the manganese reductant NH2OH as a probe to examine the function of Ca2+ in photosystem II reveals that (1) Ca2+ slows the rate of NH2OH inhibition of O2 evolution activity, but only in photosystem II membranes depleted of extrinsic proteins, (2) other divalent cations (Sr2+, Cd2+) that compete for the Ca2+ site also slow NH2OH inhibition, (3) Ca2+ is noncompetitive with respect to NH2OH, (4) in order to slow inhibition, Ca2+ must be present prior to the initiation of NH2OH reduction of manganese, and (5) Ca2+ appears not to interfere with NH2OH reduction of manganese. We conclude that the ability of Ca2+ to slow the rate of NH2OH inhibition arises from the site in photosystem II where Ca2+ normally stimulates O2 evolution and that the mechanism of this phenomenon arises from the ability of Ca2+ or certain surrogate metals to stabilize the ligation environment of the manganese complex.

Calcium↗

PubMed1989

Manganese-binding proteins of the oxygen-evolving complex.

The extrinsic 33-kDa protein (P33) was cross-linked covalently to the binding site on P33-depleted PSII preparations which is responsible for reconstitution of photosynthetic water oxidation after PSII preparations have been washed with 1 M CaCl2. Conditions were found in which more than half of the cross-linked protein complexes formed in the PSII preparations retained the ability to catalyze the oxidation of water. The complex is composed of the P33 cross-linked to the D1 and D2 proteins and a 34-kDa protein, which is present in lower abundance than the other three proteins. After solubilization of the membranes with SDS and purification by preparative SDS-PAGE, the complex retains bound manganese and can catalyze the conversion of H2O2 to O2. Calcium and chloride increased the catalase activity of the purified cross-linked complex while lanthanum or hydroxylamine abolished the activity. By use of the specific activity of the H2O2-dependent reaction to follow the extent of purification of the cross-linked complex, the most highly purified complex was determined to contain 0.34 microgram of manganese/180 micrograms of protein. The mole ratio of Mn/protein was calculated to range from 3.6 to 4.5 depending on the assumed stoichiometry of the protein subunits. The results presented here provide direct evidence that one or more of the three proteins that have cross-linked to the P33 are responsible for binding the manganese of the oxygen-evolving complex.

Catalase↗

PubMed1987

Kinetics of O2 evolution from H2O2 catalyzed by the oxygen-evolving complex: investigation of the S1-dependent reaction.

The evolution of O2 from H2O2 catalyzed by the oxygen-evolving complex (OEC) in darkness was examined with photosystem II reaction center complex preparations from spinach. Flash illumination of dark-adapted reaction centers was used to make S0-enriched or S1-enriched complexes. The membranes catalyzed O2 evolution from H2O2 when preset to either the S0 or S1 state. However, only the S0-state reaction was inhibited by carbonyl cyanide m-chlorophenylhydrazone and dependent on chloride. These results indicate that (1) the S0-dependent and S1-dependent catalytic cycles can be separated by flash illumination, (2) the S0-dependent reaction involves the formation of the S2 state, and (3) the S1-dependent reaction does not involve the formation of the S2 or S3 states. A kinetic study of the S1-dependent reaction revealed a rapid equilibrium ordered mechanism in which (1) the binding of Ca(II) must precede the binding of H2O2 to the OEC and (2) the reaction of Ca(II) with the free enzyme is at thermodynamic equilibrium such that Ca(II) does not necessarily dissociate after each catalytic cycle.

Calcium↗

PubMed1987

Hydrogen peroxide as an alternate substrate for the oxygen-evolving complex.

Photosystem II reaction centers evolve O2 in the dark when H2O2 is added as a substrate. Although some of this activity can be attributed to catalase, as much as 75% of the activity was not affected by the addition of 1 mM KCN. Several lines of evidence demonstrate that this KCN-insensitive O2 evolution from H2O2 in the dark is catalyzed by the cycling of S states in the oxygen-evolving complex including: inactivation of H2O2-mediated O2 evolution by Ca/EDTA washing; susceptibility of the activity to inhibition by amines like ammonia and Tris; inhibition by CCCP which is known to accelerate the rate of deactivation of the S2 state and; a direct dependence of the rate of O2 evolution on the presence of calcium and chloride.

Calcium↗