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M.-A. von Mach
J. G. Hengstler
Leibniz Research Centre for Working Environment and Human Factors
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L. S. Weilemann, Mainz, Germany
I. Ishibashi, Tokyo, Japan
P. Micke, Uppsala, Sweden
J. Timmer, Freiburg, Germany
A. Winterpacht, Erlangen, Germany
B. C. Behera, Pune, India

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Manganese inactivation of renal betaine aldehyde dehydrogenase from swine
(2006-10-16) Figueroa-Soto, Ciria G.; Ruíz-López, Judith C.; Valenzuela-Soto, Elisa M.
Manganese is an essential micronutrient for mammals, however high manganese concentrations cause adverse health effects. Swine renal betaine aldehyde dehydrogenase catalyzes the synthesis of glycine betaine, which plays an important role in renal cells osmoregulation. In vitro inactivation of BADH was observed by incubating the purified enzyme in the presence of 1 mM MnCl2 under physiological and low ionic strength conditions. Enzyme inactivation followed first order kinetics in a monophasic process with an inactivation constant of 0.126 ± 0.011 min^-1 and 0.137 ± 0.017 at physiological and low ionic strength, respectively. Enzyme inactivation was not prevented by physiological ionic strength, nor by the substrates NAD+ and betaine aldehyde at saturated concentrations. The enzyme was reactivated with DTT and GSH. Native-PAGE of the inactivated enzyme showed no change in the tetrameric conformation. Intrinsic protein fluorescence studies demonstrated an increased exposure of the tryptophan residues to the aqueous solvent when the enzyme was incubated with Mn^2+. These results suggest that BADH inactivation by Mn^2+ may result from the oxidation of cysteines, which induces changes in the tertiary structure of the enzyme.
A method for determining states in the course of protein unfolding
(2006-09-27) Mahdavi, Seyed Mohammad; Marashi, Sayed-Amir; Rezaei-Tavirani, Mostafa
A simple model is presented for analyzing a set of spectra obtained from spectrophotometric study of protein titration. With this model one can determine the states of (probable) intermediates in the course of protein unfolding. The model is developed based on abundance of native state, intermediate(s) and denatured state, and their contributions to differential absorbance at selected wavelengths. The model is tested for the two-state unfolding of ribonuclease A by urea in formate buffer, and also for the three-state unfolding of a-lactalbumin by guanidine hydrochloride (GdnHCl) in phosphate buffer. It was demonstrated that unfolding of ribonuclease A is matched acceptably with the two-state model, while a-lactalbumin unfolding starts with a two-state mechanism (when [GdnHCl]>=1.6M) followed by a three-state pathway.
In silico design for synthesis of molecularly imprinted microspheres specific towards bisphenol A by precipitation polymerization
(2006-09-26) Bülow, Leif; Isarankura-Na-Ayudhya, Chartchalerm; Nantasenamat, Chanin; Prachayasittikul, Virapong; Ye, Lei
Bisphenol A (BPA), a ubiquitous chemical used in industries, has attracted great attention due to its widespread leakage to the environment and foodstuff. This has spurred great interest in the preparation of synthetic polymers capable of selectively sequestering BPA. In this study, theoretical calculation was utilized to confirm the selection of suitable functional monomer capable of strong interaction with BPA. It was found that 4-vinylpyridine was the optimal functional monomer as demonstrated in the literature. A series of molecularly imprinted polymers (MIPs) were prepared by varying the functional monomer, cross-linker, and porogen. After template removal, rebinding with the original template molecule was carried out in acetonitrile, acetonitrile/water (50/50 - 20/80, v/v). 4-vinylpyridine co-polymerized with ethylene glycol dimethacrylate (4VPY-co-EDMA) and 4-vinylpyridine co-polymerized with trimethylolpropane trimethacrylate (4VPY-co-TRIM) were found to exhibit good binding performance towards bisphenol A in acetonitrile. However, only 4VPY-co-EDMA was able to maintain its imprinting effect in acetonitrile/water (50/50 v/v) whereas 4VPY-co-TRIM totally lost its imprinting effect.
Bite mark analysis in forensic routine case work
(2006-09-04) Lessig, R.; Weber, M.; Wenzel, V.
The individuality of the human dentition frequently allows the Forensic Odonto-Stomatologist (FOS) to reach a strong opinion of association in cases of identification and bite mark analy-sis. Such analysis can often be useful during the investigation of violent crimes, especially those involving sexual assault. Bites from animals are rarely the object of bite mark analysis. The teeth of animals leave patterned injuries that appear quite different from those created by human teeth. This is especially true with dogs, which are predominant culprits in bites to humans. Dogs bite humans at a rate eight times more frequently than humans bite each other. However, such bites may need to be analyzed in order to distinguish what species of animal may have been the attacker, or exclude one or more animals when there is more than one possible offender. Typical cases of routine bite mark analysis encountered by the FOS are presented. Two cases of dog bites appearing as possible accidents and two human bites report about this spectrum. In another case, a child abuse with several specific bite marks shows the potential to detect the perpetrator. The last case representing a bite mark in a fruit is obtained from criminal routine case work. It is hoped that these cases will demonstrate the significant role the analysis of bite marks might play alongside other criminalistic routines. The FOS is often involved in a late stage of the investigation. This is one reason for the problems associated with the bite mark analysis in the cases presented. Additionally, the quality of the documentation of patterned injuries is often incomplete.
Hepatotoxicity of Microcystis aeruginosa Strains Growing as Blooms in Certain Eutrophic Ponds
(2006-07-03) Jha, Prabhat N.; Kumar, Anil; Kumar, Ashok; Singh, Dhananjay P.; Sinha, Rajeshwar P.; Tyagi, Madhu B.
Critical assessment of five eutrophicated ponds of Varanasi city (India) revealed the presence of heavy blooms of cyanobacteria consisting mainly of Microcystis aeruginosa. Crude aqueous extracts of blooms as well as laboratory grown M. aeruginosa isolated from three ponds, namely Lakshmikund, Durgakund and Adityanagar showed toxicity in mouse bioassay test. Crude aqueous extracts from these samples caused death of test mice within 1h of administration (i.p.) with a LD50 of 60 mg/kg body weight and the treated animals showed clinical signs of hepatotoxicity. However such an effect was not associated with the blooms from Laatbhairov and Surajkund ponds suggesting that not all strains of M. aeruginosa are toxic. Based on spectral properties (?max 230 nm), and comparison with standard microcystin-LR, the toxin is tentatively identified as microcystin-LR. The purified toxin caused death of test mice within 40 min of its administration with a LD50 of 100 µg/ kg body weight and induced gross morphological and functional changes in liver. A 1.55 fold increase in liver weight accompanied by deep red coloration most probably due to hemorrhage and blood pooling suggested the hepatotoxic properties of the toxin. Hepatotoxicity was also evident from the drastic increase (up to 2.5 fold) in activity of serum enzymes such as glutamate pyruvate transaminase/alanine aminotransferase (GPT/ALT), lactate dehydrogenase (LDH) and alkaline phosphatase (APase) following toxin treatment. ^14C-labelling experiments demonstrated maximum accumulation (~15%) of ^14C- toxin after 20 min. of toxin administration. Appreciable level of toxin was also detected in water of four ponds. In conclusion these results clearly demonstrate that microcystin-producing blooms of M. aeruginosa are common in eutrophicated ponds of Varanasi city but not all ponds harbour toxic blooms.
Predicting networking couples for metabolic pathways of Arabidopsis
(2006-05-19) Cai, Yu-Dong; Chou, Kuo-Chen; Zhong, Wei-Zhu
Given an enzyme-compound couple, how can we identify whether it belongs to a networking couple or non-networking couple? This is very important for investigating the metabolic pathways. To address this problem, a novel approach was developed that is featured by using the knowledge of gene ontology (GO), chemical functional group (FunG), and pseudo amino acid composition (PseAA) to represent the samples of enzyme-compound couples. Two basic identifiers were formulated: one is called "GOFunG", and the other, "PseAA-FunG". The prediction was operated by fusing these two basic identifiers into one. As a showcase, the metabolic pathways were investigated for Arabidopsis thaliana, a small flowering plant widely used as a model organism for studies of the cellular and molecular biology of flowering plants. The average overall success rate via the jackknife cross-validation tests for the 72 metabolic pathways in the Arabidopsis system was over 95%, suggesting that the current approach might become a very useful tool for studying metabolic pathways and many other problems in the cellular networking related areas.
A Comparison between Low and High-dose of Hydroxyethylstarch Solution in Resuscitation for Shock induced by Ischemia/Reperfusion in Rabbits
(2006-05-19) Backer, Daniel De; Dimopoulos, George; Lobo, Suzana M.; Sun, Qinghua; Tu, Zizhi; Vincent, Jean-Louis; Xiao, Xianzhong
The purpose of the study was to compare the effects of high and low dose of 6% hydroxyethyl starch solution (HES) on resuscitation for shock induced by intestinal ischemia/reperfusion (I/R) injury in rabbits. Thirty-two anesthetized rabbits were randomized into four groups of eight animals each, which was either treated with no fluid resuscitation as control, lactated Ringer's solution (LRS, 20ml/kg/h), LRS+HES (LRS 18ml/kg/h + HES 2ml/kg/h, low dose of HES) or only treated with HES (high dose of HES, 20ml/kg/h). These rabbits underwent the intestinal I/R injury developed by occluding superior mesenteric artery (SMA) with a noncrushing vascular clamp for 60min and then loosing the clamp for 240min. The fluid resuscitation began at the same time of reperfusion. Hemodynamic parameters including MAP, HR, aortic velocity (Qaorta, as CO) and SMA blood flow (Osma) were measured. Tissue oxygenation was assessed indirectly by measuring the tonometric parameters of gut, including difference between intestinal intramucosal PtCO2 and arterial PaCO2 (PCO2-gap), intestinal intramucosal pH (pHi), arterial lactate acid concentration and oxygen delivery (DO2). Mortality of the rabbits was calculated at the end. The results showed that hemodynamic parameters were significantly higher in group LRS+HES and HES than in group LRS and control (P<0.05). Low dose of HES was better than high dose of HES in restoring hemodynamic parameters (P<0.05). Low dose of HES could greatly decrease lactate and PCO2-gap, significantly improve pHi than other three groups (P<0.05), but high dose of HES did not do so, rather, which induced oral and nasal bleeding, even death of some animals. Low dose and high dose of HES significantly improved DO2 while LRS did not (P<0.05). Therefore low dose of HSE together with LRS was more effective than only high dose of HES or LRS in the resuscitation for shock induced by intestinal I/R injury in rabbits, because hemodynamic parameters increased suitably and tissue oxygenation was greatly improved.
The effects of exogenous hormones on genetic tumor formation in Nicotiana hybrids
(2006-04-18) Heo, Seong-Il; Qu, Guan-zheng; Wang, Myeong Hyeon; Yoon, Byeong-Sung
The effect of exogenous auxin and cytokinin on cell proliferation and differentiation varies with the tissue system, and depends on the endogenous levels of the two hormones in the tissue. In this report, cytokinin (N6-benzyladenine, BA) and auxin (indole-3-acetic acid, IAA) were used to treat young seedlings, leaf explants, and genetic tumors of a Nicotiana hybrid. Exogenous BA markedly accelerated genetic tumor formation in young seedlings, while exogenous IAA inhibited it on young seedlings but not on leaf explants. Genetic tumors showed low sensitivity to exogenous BA and IAA. RAPD analysis of the genomes of the genetic tumors and normal tissue suggested that tumor formation is related to genomic instability.
Expression of interleukin-4, interleukin-9 and interleukin-13 in peripheral blood mononuclear cells of cystic fibrosis patients with and without allergy
(2006-11-25) Daigneault, Patrick; Hamid, Qutayba; Hauber, Hans-Peter; Matouk, Elias; Paque, France
Bronchial hyperresponsiveness (BHR) and mucus overproduction are common in CF pulmonary disease and allergic reactions can be frequently observed in cystic fibrosis (CF) lung disease. However, the underlying pathophysiological mechanisms are far from being completely understood. Therefore the expression of the Th2 type cytokines interleukin (IL)-4, IL-9 and IL-13 in CF patients with allergy was compared to patients without allergy. Peripheral blood mononuclear cells (PBMC) samples from 9 allergic CF patients and 8 non-allergic CF patients were obtained. In situ hybridization and immunocytochemistry were performed to determine mRNA and protein expression of IL-4, IL-9, and IL-13 in PBMC. PBMC from allergic CF patients expressed significantly more IL-13 mRNA and protein compared to non-allergic patients (p < 0.05). There was no significant difference for IL-4 and IL-9 between the two groups. In both groups IL-9 mRNA and protein expression were significantly higher compared to IL-4 and IL-13 expression (p < 0.05). These results suggest that IL-13 plays an important role in allergic disease in CF. Moreover, IL-9 may be important in CF disease whether allergy is present or not as it may contribute to BHR and mucus overproduction.
Interaction of aromatic isocyanates with n-acetyl-lcysteine under physiological conditions
(2006-11-20) Mormann, Werner; Seel, Klaus; Vaquero, Rosario Lucas
Isocyanates are of toxicological relevance since they are considered to cause occupational asthma. The majority of polyurethanes is based on aromatic diisocyanates (e.g., TDI and MDI), they are used for foams, elastomers, adhesives and coatings. Therefore we studied reactions of p-tolylisocyanate (pTI), 2,4-toluene diisocyanate (TDI) and 4,4- methylenediphenyl diisocyanate (MDI) with N-acetyl-L-cysteine (AcCys) with different molar ratios in aqueous buffer solutions of pH 5.0 - 7.4. Type and amounts of products formed in these reactions were identified and quantified. Conjugates of AcCys and the aromatic isocyanates have been synthesized and characterized as reference materials. Conjugates and ureas were found to be the main products. The ratio of these two compounds varied with the ratio of AcCys to isocyanate. Approximately 90 % of pTI conjugate were found for the 10 : 1 ratio, approximately 40 % conjugate for the 10 : 5 and around 15 % for the 10 : 15 ratio. For TDI yields of conjugate were comparable. Ureas, apart from minor amounts of TDA-urea could not be determined quantitatively due to formation of oligomeric ureas with different end groups. Minor amounts of MDI-conjugates were found apart from high amounts of insoluble material, which proved to be unreacted MDI encapsulated by oligomeric ureas. The reaction of the -SH group with the isocyanate moiety is independent of the pH of the solution in the range studied. No diamine, i.e. 2,4-TDA or 4,4-MDA, could be detected in reactions of the diisocyanates 2,4-TDI or 4,4-MDI. Small amounts of p-toluidine (pTA) were found in the reaction of the mono isocyanate pTI when it was in excess with respect to AcCys. Reactions of the isocyanates with an aqueous buffer solution of pH 6.5 in the absence of AcCys gave ureas as main products, while significant amounts of unreacted diisocyanates remained encapsulated in the mixture. No 2,4-TDA or 4,4-MDA was detected under these conditions. Again small amounts of pTA were formed from the reaction of pTI with water.
Oxaliplatin up-regulated the function and expression of P-glycoprotein/MDR1 in porcine kidney epithelial LLC-PK cells
(2006-10-26) Kishi, Hisato; Kitada, Noriaki; Ohnishi, Noriaki; Sakaeda, Toshiyuki; Takara, Kohji; Yokoyama, Teruyoshi
The purpose of this study was to clarify the effects of oxaliplatin on the function and expression of the multidrug efflux transporter P-glycoprotein/MDR1 in a porcine kidney epithelial cell line, LLC-PK, as a renal tubular epithelial model. LLC-PK cells were pretreated with or without oxaliplatin for 48 h, and the growth inhibitory effects of a MDR1 substrate, paclitaxel, and the transport of a MDR1 substrate, Rhodamine123, were assessed. The level of MDR1 mRNA and protein was also examined in the cells treated with or without oxaliplatin for 48 h using RT-PCR and immunoblotting. In the present study, the pretreatment with oxaliplatin tended to suppress the growth inhibitory effects of paclitaxel in LLC-PK cells, presumably by accelerating the functions of MDR1. In addition, the uptake of Rhodamine123 was reduced significantly by pretreatment with oxaliplatin, and the efflux of Rhodamine123 from LLC-PK cells was enhanced significantly. These accelerated functions were supported by the suppression of Rhodamine123 s transport by a representative MDR1 substrate/inhibitor, ciclosporin, at 10 µM. The exposure to oxaliplatin for 48 h resulted in an increase in the expression of MDR1 in LLC-PK cells. These findings were similar to those obtained with cisplatin, a nephrotoxic drug. In conclusion, the present findings suggested that transient exposure for 48 h to oxaliplatin caused the up-regulation of MDR1 function and expression in LLC-PK cells, as was the case for cisplatin.
Insights into the genetic re-engineering of chimeric antibody-binding green fluorescent proteins for immunological taggers
(2006-10-16) Bülow, Leif; Isarankura-Na-Ayudhya, Chartchalerm; Prachayasittikul, Virapong; Suwanwong, Yaneenart
Genetic re-manipulation of chimeric antibody-binding green fluorescent proteins was successfully conducted to create versatile tools for immunological diagnosis. Four chimeric GFPs carrying one and two-consecutive sequences of the Fc-binding motif (Z-domain), derivative of IgG-binding B domain of Staphylococcal protein A (SpA), at the C-terminus were constructed. The chimeric Ab-binding GFPs possessed dual characteristics of both IgG-binding and activity of fluorescent emission. The chimeric proteins were purified to homogeneity using an IgG-Sepharose column. Additionally, a hexahistidine was fused to the N-terminal of the GFPZ and GFPZZ to allow a high protein recovery obtained from immobilized metal (Ni^2+) affinity chromatography (Ni-NTA), and for protein immobilization to the sensor surface. Results obtained from the Surface Plasmon Resonance (SPR) revealed a high binding affinity (K_a) to immobilized human immunoglobulin up to 6.7 and 81.1 (107/M) for the GFPZ and GFPZZ, respectively. This affinity constant was raised up to 2-5 times higher when the chimeric GFPs harboring hexahistidine residues were captured on the sensor chip via metal coordination. The strong binding affinity to IgG of the chimeric GFPs was clinically applied to detect the antinuclear antibody. A strong intensity of fluorescence, higher than that of the classical fluorescein isothiocyanate (FITC) conjugated system, was significantly detected. Moreover, the proteins with double repeats of Fc-binding motif (GFPZZ and H6GFPZZ) obviously demonstrated a more intense fluorescent signal than those of the single Z domain, which corresponded to the result from SPR. All these findings support a high potential for applying such chimeric Ab-binding GFPs for clinical applications.
Prediction of selectivity index of pentachlorophenol-imprinted polymers
(2006-10-16) Isarankura-Na-Ayudhya, Chartchalerm; Naenna, Thanakorn; Nantasenamat, Chanin; Prachayasittikul, Virapong; Tantimongcolwat, Tanawut
A data set comprising of the selectivity index of pentachlorophenol-imprinted polymers against 53 pentachlorophenol and related compounds was obtained from the excellent work of Baggiani et al. Molecular descriptors of the phenol compounds were calculated with E-DRAGON to obtain a total of 1,666 descriptors spanning 20 categories of molecular properties. Multivariate analysis of the data set was performed using multiple linear regression, partial least squares regression, and principal component regression. Partial least squares regression was found to deliver an excellent predictive model and was chosen for further investigation. The descriptor dimension was reduced by the combined use of partial least squares and Unsupervised Forward Selection algorithm. The obtained Quantitative Structure-Property Relationship (QSPR) model based on the smaller subset of the molecular descriptors displayed substantial gain in predictive ability when compared to the model of Baggiani et al. Such QSPR model can help in the computational design of MIPs with predefined selectivity toward template molecules of interest.
Genetic polymorphism of Clara cell secretory protein 16KDa (CC16) and susceptibility to asthma, a meta-analysis
(2006-01-05) Saadat, Mostafa
Clara cell secretory protein 16KDa (CC16) is the major secretory protein of the Clara cells. The gene encoding CC16 has been implicated as potential susceptibility gene for asthma. Published studies between genetic polymorphism of CC16 at position 38 and asthma risk are controversial. Most of these studies are based on small sample size. To clarify the role of the A38G CC16 polymorphism on the risk of developing asthma, we carried out a meta-analysis of the research published between January 1988 and August 2005. In the present study, 11 case-control studies with 2362 subjects (1262 controls and 1100 patients) were eligible for meta-analysis. The fixed-effects method was used when there was no significant heterogeneity between the results of the individual studies being pooled, whereas, the random-effects method was used when there was heterogeneity between the studies. The overall ORs of the asthma risk were not associated with the CC16 genotypes (For AG vs GG: OR=1.09, 95% CI: 0.90-1.32; For AA vs GG: OR=1.05, 95% CI: 0.79-1.39; For AG+AA vs GG: OR=1.09, 95% CI: 0.91-1.30). It should be noted that similar results were obtained when we used the stratified data, adjusted for age and smoking status of the subjects. In conclusion, the CC16 A38G polymorphism was not associated with asthma risk in the present meta-analysis.

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