Lysine 194 Is Functional in Isocitrate Lyase from Escherichia coli

Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had k_cat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. K _m values for Mg²⁺-D_s-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased K _m value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state. Received: 3 December 1996 / Accepted: 18 December 1996     

Serine319 and 321 Are Functional in Isocitrate Lyase from Escherichia coli

With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichia coli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the k_cat of E. coli isocitrate lyase relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05; S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and S319A/S321A, 0, with little or no effect on the K _m for the substrate Mg²⁺-D_s-isocitrate. The most active variant S319T exhibited threefold less activity than the wild-type enzyme; all variants assembled into tetramers. The S319T mutant isocitrate lyase was 100-fold more active than the S321T variant. This observation suggests that the requirement for a β-hydroxymethyl group of serine in catalysis is less important at position 319 than at position 321. Although most singly substituted variants had very low isocitrate lyase activity, all variants harboring mutant isocitrate lyase of very low activity did grow on acetate as a sole carbon source albeit with longer doubling times and lag phases. Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to activity and increased the K _m for substrate 3.5- to 8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups and facilitates substrate binding and catalysis. From these collective results, it is proposed that Ser319 and Ser321 are involved in E. coli isocitrate lyase catalysis, perhaps by stabilizing the postulated reaction intermediate succinate trianion in the aci-carboxylate form and the related transition state via hydrogen bonding. Received: 3 September 1996 / Accepted: 20 September 1996     

Isomeric acetoxy analogs of celecoxib and their evaluation as cyclooxygenase inhibitors

A group of celecoxib analogs having a SO(2)NH(2) (9a-f), or SO(2)Me (12a-f), COX-2 pharmacophore at the para-position of the N-1 phenyl ring in conjunction with a C-5 phenyl ring having a variety of substituents (4-, 3-, 2-OAc; 4-Me,2-OAc, 4-Me,3-OAc, 4-F,2-OAc) was synthesized for evaluation as cyclooxygenase (COX) inhibitors of the COX-1/COX-2 isozymes. Within this group of compounds, 1-(4-aminosulfonylphenyl)-3-trifluoromethyl-5-(2-acetoxy-4-fluorophenyl)pyrazole (9f) emerged as the most potent (COX-1 IC(50)=0.7 μM; COX-2 IC(50)=0.015 μM) and selective (COX-2 selectivity index=47) inhibitor agent that exhibited good anti-inflammatory activity (ED(50)=42.3mg/kg) which was lower than the reference drug celecoxib (ED(50)=10.8 mg/kg), but greater than ibuprofen (ED(50)=67.4 mg/kg) and aspirin (ED(50)=128.7 mg/kg). Molecular modeling studies for 9f showed that the SO(2)NH(2) group assumes a position within the secondary pocket of the COX-2 active site wherein the SO(2)NH(2) oxygen atom is hydrogen bonded to the H90 residue (2.90?), the SO(2)NH(2) nitrogen atom forms a hydrogen bond with L352 (N?O=2.80?), and the acetyl group is positioned in the vicinity of the S530 residue where the acetyl oxygen atom undergoes hydrogen bonding to L531 (2.99?).     

Mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress and improve insulin secretion in glucotoxicity and glucolipotoxicity

Mitochondrial oxidative damage is thought to play a key role in pancreatic β-cell failure in the pathogenesis of type 2 diabetes. Despite this, the potential of mitochondria-targeted antioxidants to protect pancreatic β-cells against oxidative stress has not yet been studied. Therefore, we investigated if mitochondria-targeted antioxidants protect pancreatic β-cells such as RINm5F and HIT-T15 cells against oxidative stress under glucotoxic and glucolipotoxic conditions. When β-cells were incubated under these conditions, the expression levels of mitochondrial electron transport chain complex subunits, mitochondrial antioxidant enzymes (such as MnSOD and Prx3), β-cell apoptosis, lipogenic enzymes (such as ACC, FAS and ABCA1), intracellular lipid accumulation, oxidative stress, ER stress, mitochondrial membrane depolarization, nuclear NF- κB and sterol regulatory element binding protein 1c (SREBP1c) were all increased, in parallel with decreases in intracellular ATP content, citrate synthase enzymatic activity and glucose-stimulated insulin secretion. These changes were consistent with elevated mitochondrial oxidative stress, and incubation with the mitochondria-targeted antioxidants, MitoTempol or Mitoquinone (MitoQ), prevented these effects. In conclusion, mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress, promote their survival, and increase insulin secretion in cell models of the glucotoxicity and glucolipotoxicity associated with Type 2 diabetes.     

Interrupted 2'-o,4'-C-aminomethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'- BNA(NC)) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2',4'-BNA(NC) modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2',4'-BNA(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2',4'-BNA(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.     

Proteomic Profiling of the Aleurone Layer of Mature Arabidopsis thaliana Seed

The aleurone layer of mature Arabidopsis thaliana seed plays important roles in seed germination and dormancy. However, the proteomic profile of this cell layer is unknown partly because it is difficult to separate this thin cell layer from the mature seeds. In this study, we have used a simple technique to separate the aleurone layer along with the seed coat following germination of seeds and determined for the first time the putative protein composition of this cell layer. By subjecting the total proteins extracted from the seed coat to 2D gel electrophoresis followed by liquid chromatography/tandem mass spectrometry, we identified four AGI loci, AT4G28520, AT5G44120, AT1G03880, and AT1G03890; all of which belong to the seed storage family of proteins. Because in Arabidopsis the diploid aleurone cells of the seed coat perform protein storage functions similar to that of triploid endosperm of other plant species, it is assumed that the above AGI loci are associated with the aleurone layer of the seed coat.     

A putative <Emphasis Type="Italic">NEM1</Emphasis> homologue regulates lipid droplet biogenesis <Emphasis Type="Italic">via PAH1</Emphasis> in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.     

Spatial distribution of toxic metals in drinking water sources and their associated health risk in district buner,Northern Pakistan

Vigorous knowledge on the occurrence and distribution of toxic metals (TMs) in theenvironment is needed to assess their toxicological impacts on human health especially in developing countries like Pakistan. For the first time, the concentrations of TMs like Ni, Zn, Pb, Cr, and Cd in both ground and surface water and their potential health risk in the district Buner (Pakistan) were determined using Inductive Coupled Plasma Mass Spectrometry (ICP-MS, Agilent Technologies, 7500 CX, USA) and their results were compared with their safe limits defined by Pakistan Environmental Protection Agency (Pak-EPA) and World Health Organization WHO (2008 WHO (World Health Organization). 2008. Guidelines for drinking water quality, First Addendum to third ed. Recommendations, vol 1. Geneva, Switzerland [Google Scholar]). The concentrations of TMs, like Pb (43% and 86%), Ni (63% and 32%), Cr (41%), and Cd (1% and 20%) in drinking water samples exceeded their respective permissible limits set by Pak-EPA and WHO (2008), respectively. The mean concentrations of TMs were found in decreasing order Ni > Pb > Cr > Cd > Zn. The HRI values of TMs in drinking water samples for both adults and children were observed <1 indicating no health risk to the local population. Excessive marble industries are present in the study area; therefore, it is necessary to reduce metal contamination via proper disposal and treatment of marble wastewater, for which the government should take serious action in the study area.     

Willingness-to-Pay for Community-Based Health Insurance among Informal Workers in Urban Bangladesh

Introduction

Reliance on out-of-pocket payment for healthcare may lead poor households to undertake catastrophic health expenditure, and risk-pooling mechanisms have been recommended to mitigate such burdens for households in Bangladesh. About 88% of the population of Bangladesh depends on work in the informal sector. We aimed to estimate willingness-to-pay (WTP) for CBHI and identify its determinants among three categories of urban informal workers rickshaw-pullers, shopkeepers and restaurant workers.

Methods

The bidding game version of contingent valuation method was used to estimate weekly WTP. In three urban locations 557 workers were interviewed using a structured questionnaire during 2010 and 2011. Multiple-regression analysis was used to predict WTP by demographic and household characteristics, occupation, education level and past illness.

Results

WTP for a CBHI scheme was expressed by 86.7% of informal workers. Weekly average WTP was 22.8 BDT [Bangladeshi Taka; 95% confidence interval (CI) 20.9–24.8] or 0.32 USD and varied significantly across occupational groups (p = 0.000) and locations (p = 0.003). WTP was highest among rickshaw-pullers (28.2 BDT or 0.40 USD; 95% CI: 24.7–31.7), followed by restaurant workers (20.4 BDT 0.29 USD; 95% CI: 17.0–23.8) and shopkeepers (19.2 BDT or 0.27 USD; 95% CI: 16.1–22.4). Multiple regression analysis identified monthly income, occupation, geographical location and educational level as the key determinants of WTP. WTP increased 0.196% with each 1% increase in monthly income, and was 26.9% lower among workers with up to a primary level of education versus those with higher than primary, but less than one year of education.

Conclusion

Informal workers in urban areas thus are willing to pay for CBHI and socioeconomic differences explain the magnitude of WTP. The policy maker might think introducing community-based model including public-community partnership model for healthcare financing of informal workers. Decision making regarding the implementation of such schemes should consider worker location and occupation.