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1.  Diabetic factors associated with gastrointestinal symptoms in patients with type 2 diabetes 
AIM: To determine whether gastrointestinal (GI) symptoms are more frequent in type 2 diabetic patients and to examine which diabetic factors are associated with the symptoms.
METHODS: Consecutive subjects with diabetes and age-/gender-matched normal controls were recruited for this study. GI symptoms were assessed using a structured questionnaire divided into two GI symptom categories (upper and lower GI symptoms), and consisting of 11 individual symptoms. In the diabetic patient group, diabetic complications including peripheral neuropathy, nephropathy and retinopathy, glycosylated hemoglobin (HbA1c) level and diabetes duration were evaluated.
RESULTS: Among the total 190 diabetic patients and 190 controls enrolled, 137 (72%) of the diabetic patients and 116 (62%) of the controls had GI symptoms. In the diabetic patient group, 83 (43%) had upper GI symptoms and 110 (58%) lower GI symptoms; in the control group, 59 (31%) had upper GI symptoms and 104 (55%) lower GI symptoms. This difference between the two groups was significant for only the upper GI symptoms (P = 0.02). Among the diabetic factors, the HbA1c level was the only independent risk factor for upper GI symptoms in the multiple logistic regression analysis (odds ratio = 2.01, 95% confidence interval: 1.02-3.95).
CONCLUSION: Type 2 diabetes was associated with an increased prevalence of upper GI symptoms and these symptoms appeared to be independently linked to poor glycemic control, as measured by the HbA1c levels.
PMCID: PMC2852829  PMID: 20380013
Diabetes; HbA1c; Upper gastrointestinal symptoms
2.  Retraction: Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing 
PLoS ONE  2013;8(5):10.1371/annotation/1345f9b0-016e-4123-9e72-6ccc4fa17ba2.
PMCID: PMC3655197  PMID: 23690906
3.  Retraction: Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing 
PLoS ONE  2013;8(4):10.1371/annotation/8f2ddf91-3499-4627-9a91-449b78465f9d.
PMCID: PMC3634625  PMID: 23853689
4.  Complete Solubilization and Purification of Recombinant Human Growth Hormone Produced in Escherichia coli 
PLoS ONE  2013;8(2):e56168.
High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.
PMCID: PMC3567055  PMID: 23409149
5.  Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing 
PLoS ONE  2012;7(5):e37102.
Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-l-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria.
PMCID: PMC3358330  PMID: 22629354
6.  Synthesis of “clickable” acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation 
Alkynyl- and azido-tagged 3-oxo-C12-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C12-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative.
PMCID: PMC3081916  PMID: 21190852
Acylhomoserine lactones; Click chemistry; Pseudomonas aeruginosa; Quorum sensing
7.  Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification 
PLoS ONE  2011;6(12):e28661.
Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, −2.1%, and −13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.
PMCID: PMC3235147  PMID: 22174862
8.  Protein Crystal Engineering of YpAC-IV using the Strategy of Excess Charge Reduction 
Crystal growth & design  2009;9(8):3570-3574.
The class IV adenylyl cyclase from Yersinia pestis has been engineered by site-specific mutagenesis to facilitate crystallization at neutral pH. The wild-type enzyme crystallized only below pH 5, consistent with the observation of a carboxyl-carboxylate H bond in a crystal contact in the refined structure 2FJT. Based on that unliganded structure at 1.9 Å resolution, two different approaches were tested with the goal of producing a higher-pH crystal needed for inhibitor complexation and mechanistic studies. In one approach, Asp 19, which forms the growth-limiting dicarboxyl contact in wild-type triclinic crystals, was modified to Ala and Asn in hopes of relieving the acid-dependence of that crystal form. In the other approach, wild-type residues Met 18, Glu 25, and Asp 55 were (individually) changed to lysine to reduce the protein's excess negative charge in hopes of enabling growth of new, higher-pH forms. These 3 sites were selected based on their high solvent exposure and lack of intraprotein interactions. The D19A and D19N mutants had reduced solubility and did not crystallize. The other 3 mutants all crystallized, producing several new forms at neutral pH. One of these forms, with the D55K mutant, enabled a product complex at 1.6 Å resolution, structure 3GHX. This structure shows why the new crystal form required the mutation in order to grow at neutral pH. This approach could be useful in other cases where excess negative charge inhibits the crystallization of low-pI proteins.
PMCID: PMC2758785  PMID: 20160955
crystal growth; crystal contact; crystal engineering; isoelectric point; mutation; structure; x-ray diffraction
9.  Crystallization of the class IV adenylyl cyclase from Yersinia pestis  
The class IV adenylyl cyclase from Y. pestis has been crystallized in an orthorhombic form suitable for structure determination.
The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 Å, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 Å, α = 88.7, β = 82.5, γ = 65.5°. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P212121. These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 Å, diffract to 3 Å and probably have two dimers per asymmetric unit and V M = 3.0 Å3 Da−1. Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.
PMCID: PMC2197185  PMID: 16511301
adenylyl cyclase; cyclic AMP; selenomethionine
10.  Biochemical and Structural Characterization of the Secreted Chorismate Mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ Enzyme Not Regulated by the Aromatic Amino Acids 
Journal of Bacteriology  2006;188(24):8638-8648.
The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M. tuberculosis. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a Km of 0.5 ± 0.05 mM and a kcat of 60 s−1 per active site (at 37°C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 Å resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.
PMCID: PMC1698256  PMID: 17146044

Results 1-10 (10)