• Journal of Microbiology and Biotechnology
• /
• v.9 no.6
• /
• pp.757-763
• /
• 1999

The metal specificity of D-xylose isomerase from Streptomyces rubiginosus was examined by site-directed mutagenesis. The activation constants for metal ion ($Mg^{2+},{\;}Mn^{2+},{\;}or{\;}Co^{2+}$) of wild-type and mutant enzymes were determined by titrating the metal ion-free enzyme with $Mg^{2+},{\;}Mn^{2+},{\;}and{\;}Co^{2+}$, respectively. Substitutions of amino acids either on coordinated or around the M2 site (His-22O, Asn-185, Glu-186, and Glu-221) dramatically affected the activation constants as well as activity. A decrease of metal binding affinity was most significant in the presence of $Mg^{2+}$. When compared with the wild-type enzymes, the binding affinity of H220S and Nl85K for Mg^{2+} was decreased by 10-15-fold, while the affinity for $Mn^{2+}{\;}or{\;}Co^{2+}$ only decreased by 3-5-fold. All the mutations close to the M2 site changed their metal preference from $Mg^{2+}{\;}to{\;}Mn^{2+}{\;}or{\;}Co^{2+}$. These altered metal preferences may be caused by a relatively weak binding affinity of $Mg^{2+}$ to the enzyme. Thermal inactivation studies of mutants at the M2 site also support the importance of the M2 site geometry for metal specificity as well as the thermostability of the enzyme. Mutations of other important groups hardly affected the metal preference, although pronounced effects on the kinetic parameters were sometimes observed. This study proposes that the metal specificity of D-xylose isomerase can be altered by the perturbation of the M2 site geometry, and that the different metal preference of Group I and GroupII D-xylose isomerases may be caused by nonconserved amino acid residues around the M2 site.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.5
• /
• pp.681-688
• /
• 2020

Bacterial cell-based biosensors, or whole-cell bioreporters (WCBs), are an alternative tool for the quantification of hazardous materials. Most WCBs share similar working mechanisms. In brief, the recognition of a target by sensing domains induces a biological event, such as changes in protein conformation or gene expression, providing a basis for quantification. WCBs targeting heavy metal(loid)s employ metalloregulators as sensing domains and control the expression of genes in the presence of target metal(loid) ions, but the diversity of targets, specificity, and sensitivity of these WCBs are limited. In this study, we genetically engineered the metal-binding loop (MBL) of ZntR, which controls the znt-operon in Escherichia coli. In the MBL of ZntR, three Cys sites interact with metal ions. Based on the crystal structure of ZntR, MBL sequences were modified by site-directed mutagenesis. As a result, the metal-sensing properties of WCBs differed depending on amino acid sequences and the new selectivity to Cr or Pb was observed. Although there is room for improvement, our results support the use of currently available WCBs as a platform to generate new WCBs to target other environmental pollutants including metal(loid)s.

• Toxicological Research
• /
• v.23 no.1
• /
• pp.1-9
• /
• 2007

Transporters are membrane proteins that mediate the transfer of substrate across the cellular membrane. In this overview, the characteristics and the toxicological relevance were discussed for various types of transporters. For drug transporters, the overview focused on ATP-binding cassette transporters and solute carrier family 21A/22A member transporters. Except for OCTN transporters and OATP transporters, drug transporters tend to have broad substrate specificity, suggesting drug-drug interaction at the level of transport processes (e.g., interaction between methotrexate and non-steroidal anti-inflammatory agents) is likely. For metal transporters, transporters for zinc, copper and multiple metals were discussed in this overview. These metal transporters have comparatively narrow substrate specificity, except for multiple metal transporters, suggesting that inter-substrate interaction at the level of transport is less likely. In contrast, the expressions of the transporters are often regulated by their substrates, suggesting cellular adaptation mechanism exists for these transporters. The drug-drug interactions in drug transporters and the cellular adaptation mechanisms for metal transporters are likely to lead to alterations in pharmacokinetics and cellular metal homeostasis, which may be linked to the development of toxicity. Therefore, the transporter-mediated alterations may have toxicological relevance.

• Imaging Science in Dentistry
• /
• v.45 no.3
• /
• pp.147-151
• /
• 2015

Purpose: The aim of this study was to investigate the effects of metal artifacts on the accurate diagnosis of root fractures using cone-beam computed tomography (CBCT) images with large and small/limited fields of view (FOVs). Materials and Methods: Forty extracted molar and premolar teeth were collected. Access canals were made in all teeth using a rotary system. In half of the teeth, fractures were created by the application of mild pressure with a hammer. The teeth were then randomly put into a wax rim on an acryl base designed in the shape of a mandible. CBCT scans were obtained using a Newtom 5G system with FOVs of $18cm{\times}16cm$ and $6cm{\times}6cm$. A metal pin was then placed into each tooth, and CBCT imaging was again performed using the same fields of view. All scans were evaluated by two oral and maxillofacial radiologists. The specificity, sensitivity, positive predictive value, negative predictive value, and likelihood ratios (positive and negative) were calculated. Result: The maximum levels of sensitivity and specificity (100% and 100%, respectively) were observed in small-volume CBCT scans of teeth without pins. The highest negative predictive value was found in the small-volume group without pins, whereas the positive predictive value was 100% in all groups except the large-volume group with pins. Conclusion: The specificity of CBCT decreased with the presence of a pin in the large-volume group, but not in the small-volume group.

• Imaging Science in Dentistry
• /
• v.54 no.1
• /
• pp.49-56
• /
• 2024

Purpose: This study evaluated the impact of artifacts generated by metal crowns on the detection of proximal caries lesions in teeth at various distances using cone-beam computed tomography (CBCT). Additionally, the diagnostic impacts of tube current and metal artifact reduction (MAR) were investigated. Materials and Methods: Thirty teeth were arranged within 10 phantoms, each containing 1 first premolar, 1 second premolar, and 1 second molar. A sound first molar (for the control group) or a tooth with a metal crown was placed. Of the 60 proximal surfaces evaluated, 15 were sound and 45 exhibited enamel caries. CBCT scans were acquired using an OP300 Maxio unit (Instrumentarium, Tuusula, Finland), while varying the tube current (4, 8, or 12.5 mA) and enabling or disabling MAR. Five observers assessed mesial and distal surfaces using a 5-point scale. Multi-way analysis of variance was employed for data comparison, with P<0.05 indicating statistical significance. Results: The area under the curve (AUC) varied from 0.40 to 0.60 (sensitivity: 0.28-0.45, specificity: 0.44-0.80). The diagnostic accuracy was not significantly affected by the presence of a metal crown, milliamperage, or MAR(P>0.05). However, the overall AUC and specificity were significantly lower for surfaces near a crown (P<0.05). Conclusion: CBCT-based caries detection was not influenced by the presence of a metal crown, variations in milliamperage, or MAR activation. However, the diagnostic accuracy was low and was further diminished for surfaces near a crown. Consequently, CBCT is not recommended for the detection of incipient caries lesions.

• Bulletin of the Korean Chemical Society
• /
• v.17 no.10
• /
• pp.905-908
• /
• 1996

A substrate specificity of cabbage phospholipase D (PLD) was studied using the synthetic phospholipids having different head groups. The phospholipids were synthesized from phosphatidylcholine and appropriate bases by transphosphatidylation of PLD. The bases used were ethanolamine, serine, ethanol and γ-hydroxybutyric acid. The phosphatidic acid, the product of PLD, was separated in TLC and measured densitometrically. The kinetic parameters were estimated for each substrate and the effects of pH, SDS, Ca2+ and other metal ions were examined. Vmax values found were 3.75, 2.36, 5.59, 1.63, 2.30 nmol/min/μg protein for phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylethanol, and phosphatidylburytic acid, respectively. These results indicate a broad specificity of cabbage PLD toward phospholipids with different head groups. Particularly phosphatidylserine was most easily hydrolyzed by PLD and its activity did not depend on Ca2+.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.9 no.1
• /
• pp.59-73
• /
• 1980

A detailed procedure was described for the isolation of cratine kinase (ATP-Creatine phosphotransferase, E. C. 2. 7. 3. 2.) from the muscle of the snake Bungarus fasciatus. The original isolation procedure of Kuby et al. for the rabbit muscle enzyme has been modified and extended to include a chromatographic step. The properties of the enzyme have been investigated and kinetic constants for the reverse reactions determined as the followings: 1) A molecular weight of the enzyme was determined by gel filteration on Sephadex G-100 and by electrophoresis on SDS-polyacrylamide was 86,000. 2) Two reactive sulphydryl groups were detected with dithiobis nitrobenzoic acid (DTNB). 3) The nucleotide substrate specificity in the reverse reaction was determined as ADP*2'-dADP＞GDP＞XDP＞UDP with magnesium as the activating metal ion. 4) The order of the metal specificity in the reverse reaction Mg>Mn>$Ca{\sim}Co$ was determined with ADP as substrate. 5) A detailed kinetic analysis was carried out in the reverse direction with $MaADP^-$ as the nucleotide substrate. Initial velocity and product inhibition studies($MaADP^{2-}$ competitive with respect to MgADP- and noncompetitive with respect to $N-phosphorycreatine^{2-}$ ; Creatine competitive with respect to $N-phosphorycreatine^{2-}$ and noncompetitive with respect to Ma $ADP^-)$ indicated that the reaction obeyed a sequential mechanism of the rapid equilibrium random type.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.2
• /
• pp.166-169
• /
• 2012

In several biotechnological applications of living bacterial cells with inducible gene expression systems, the extent of overexpression and the specificity to the inducer are key elements. In the present study, we established the concentration ranges of $Zn^{2+}$, $Ni^{2+}$, $Co^{2+}$, ${AsO_2}^-$, and $Cd^{2+}$ ions that caused significant activation of the respective promoters of Synechocystis sp. without concomitant unspecific stress responses. The low expression levels can be increased up to 10-100-fold upon treatments with $Cd^{2+}$, ${AsO_2}^-$, $Zn^{2+}$, and $Co^{2+}$ ions and up to 800-fold upon $Ni^{2+}$ treatment. These results facilitate the development of conditional gene expression systems in cyanobacteria.

• Proceedings of the Korean Society for Bioinformatics Conference
• /
• 2003.10a
• /
• pp.5-5
• /
• 2003

The purple acid phosphatases comprise a family of binuclear metal-containing enzymes. The metal centre contains one ferric ion and one divalent metal ion. Spectroscopic studies of the monomeric, ${\sim}$36 kDa mammalian purple acid phosphatases reveal the presence of an Fe(III)Fe(II) centre in which the metals are weakly antiferromagnetically coupled, whereas the dimeric, ${\sim}$110 000 kDa plant enzymes contain either Fe(III)Zn(II) or Fe(III)Mn(II). The three dimensional structures of the red kidney bean and pig enzymes show very similar arrangements of the metal ligands but some significant differences beyond the immediate vicinity of the metals. In addition to the catalytic domain, the plant enzyme contains a second domain of unknown function. A search of sequence databases was undertaken using a sequence pattern which includes the conserved metal-binding residues in the plant and animal enzymes. The search revealed the presence in plants of a 'mammalian-type' low molecular weight purple acid phosphatase, a high molecular weight form in some fungi, and a homologue in some bacteria. The catalytic mechanism of the enzyme has been investigated with a view to understanding the marked difference in specificity between the Fe-Mn sweet potato enzyme, which exhibits highly efficient catalysis towards both activated and unactivated phosphate esters, and other PAPs, which hydrolyse only activated esters. Comparison of the active site structures of the enzymes reveal some interesting differences between them which may account for the difference. The implications fur understanding the physiological functions of the enzymes will be discussed.

• Journal of Acupuncture Research
• /
• v.26 no.5
• /
• pp.11-17
• /
• 2009

Objectives : The objectives of this study were to examine the specificity and the sensitivity of tenderness testing on the ear about the postpartum breast pain, and to improve the efficacy of the tenderness test. Methods : Thirty women who visited in the hospital for recuperating 2-14 days after childbirth participated in this study. They answered the questionnaire of the severity of breast pain and their auricular acupuncture points were tested by a long metal probe with round tip 1.1mm in diameter. We analyzed the relationship between auricular tenderness testing on mammary gland zone[CW6] and postpartum breast pain with validity and significance as grouping 8 diagnosis methods. Results : When we consider above 'moderate pain' or 'severe pain' in the auricular tenderness testing as a positive sign, and above 'evere pain' or 'very severe pain' in the self-reported breast pain score as a positive symptom, the diagnostic efficacy was highest. The sensitivity for tenderness testing was 0.62-0.75, and the specificity was 0.64-0.72, and the sensitivity and the specificity in the contralateral ear were a little higher than in the ipsilateral ear. Conclusions : These results suggest that auricular tenderness testing can be objectified clinically and be used in diagnosis.