Identification of alpha-bungarotoxin (A31) as the major postsynaptic neurotoxin, and complete nucleotide identity of a genomic DNA of Bungarus candidus from Java with exons of the Bungarus multicinctus alpha-bungarotoxin (A31) gene.

The Malayan krait (Bungarus candidus) is one of the most medically significant snake species in Southeast Asia. No specific antivenom exists to treat envenoming by this species. Death within 30 min after its bite has been reported from Java, suggesting the presence of highly lethal postsynaptic neurotoxins in the venom of these snakes. We purified and identified the major postsynaptic toxin in the venom of B. candidus from Java. The toxin was indistinguishable from alpha-bungarotoxin (A31), a toxin originally isolated from Bungarus multicinctus, in its mass (7983.75 Da), LD50 (0.23 microg/g in mice i.p.), affinity to nicotinic acetylcholine receptors, and by its 40 N-terminal amino acid residues as determined by Edman degradation. Identity with alpha-bungarotoxin was confirmed by cloning and sequencing a genomic DNA from B. candidus which encodes the 74 amino acid sequence of alpha-bungarotoxin (A31) and part of its signal peptide, revealing complete identity to the alpha-bungarotoxin (A31) gene in exon and 98.9% identity in intron sequences. The entire mitochondrial cytochrome b gene of the krait species B. candidus from Java and B. multicinctus from Taiwan was sequenced for comparison, suggesting that these snakes are phylogenetically closely related. alpha-Bungarotoxin appears to be widely present and conserved in Southeast and East Asian black-and-white kraits across populations and taxa.     

6-(1-Alkenoyloxyalkyl)-5,8-dimethoxy-1,4-naphthoquinone derivatives: Synthesis and evaluation of antitumor activity

Thirty six 5,8-dimethoxy-1,4-naphthoquinone derivatives, which bear unsaturated alkyl side chain with ester bond, were synthesized and tested cytotoxic activity on L1210 cells and antitumor activity against ICR mice bearing S-180 cells. It could be recognized that the cytotoxicities of naphthoquinones with R₁ being methyl and propyl (IV1∼24) were not enhanced by replacing the alkanoyls with alkenoyls. In contrast, the introduction of the alkenoyl moieties on the compounds with R₁=hexyl (IV25∼36) resulted in the enhancement of their cytotoxicities. Replacement of alkanoyl group with an alkenoyl group generally increased the T/C value of the mice bearing S-180 cells.     

Glucose-Responsive Gel from Phenylborate Polymer and Poly (Vinyl Alcohol): Prompt Response at Physiological pH Through the Interaction of Borate with Amino Group in the Gel

Purpose. To design glucose-responsive gels based on the complexation between polymers having phenylboronic acid groups and poly (vinyl alcohol). Specifically, high-glucose sensitivity at physiological pH was achieved through the interaction of phenylborate with amino groups. Method. Terpolymers of m-acrylamidophenylboronic acid, N,N-dimethylaminopropylacrylamide (DMA-PAA), and N,N-dimethylacrylamide were prepared. DMAPAA was introduced in the terpolymer to stabilize phenylborate-polyol complex at physiological pH. The effect of amino groups on complex stabilization was estimated from viscosity as well as UV spectrum measurements. Results. A good correlation was observed between complexation rate and fraction of phenylborate as well as DMAPAA in the terpolymers. In line with this increased complexation rate. UV difference spectra measurement revealed that ionization of phenylboronic acid was facilitated in the terpolymer due to the interaction with DMAPAA. Further, sensitive change in the complexation rate was demonstrated with a variation in glucose concentration, which is in sharp contrast with the poor glucose-sensitivity of the polymer without DMAPAA. Conclusions. The introduction of an amino group into phenylborate polymers was quite effective for increasing the complexation ability and the glucose responsivity at physiological pH. These results suggest the feasibility of this complex-gel system in designing a self-regulated insulin-releasing device.     

Investigation of the potential for binding of di(2-ethylhexyl) phthalate (DEHP) and di(2-ethylhexyl) adipate (DEHA) to liver DNA in vivo

It was the aim of this investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or without pretreatment for 4 weeks with 1% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [¹⁴C]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The limit of detection, CBI < 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [¹⁴C]- and [³H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where the carcinogen-modified nucleoside or base adducts are expected. The respective limits of detection were at 0.07 and 0.04 CBI units for the ¹⁴C and ³H labels, respectively. The experiments with [¹⁴C]- and [³H]DEHA, labeled in the alcohol moiety and administered to mice, revealed a minute radioactivity of <50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which had been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for ¹⁴C and 0.01 to 0.12 for ³H resulted. Determination of the level of ¹⁴CO₂ expiration revealed a linear correlation with the specific activity of DNA. Experiments with 2-ethyl[1-¹⁴C]hexanol performed with both rats and mice allowed the conclusion that most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01. Pretreatment of the animals with unlabeled compound had no effect on the DNA radioactivities in either species. The present negative data, in conjunction with other negative short-term tests for mutagenicity, strongly indicate that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP and DEHA in rodents.     

Identification of Major Esterase Involved in Hydrolysis of Soft Anticholinergic (2R3’R-SGM) Designed From Glycopyrrolate in Human and Rat Tissues

The glycopyrrolate soft analog, SGM, designed to be easily hydrolyzed into the significantly less active zwitterionic metabolite, SGa, typifies soft drug that reduces systemic side effects (a problem often seen with traditional anticholinergics) following local administration. In this study, hydrolysis of 2R3’R-SGM, the highest pharmacologically active stereoisomer of SGM, was investigated in human and rat tissues. In both species, 2R3’R-SGM was metabolized to 2R3’R-SGa in plasma but was stable in liver and intestine. The half-life of 2R3’R-SGM was found to be 16.9 min and 9.8 min in human and rat plasma, respectively. The enzyme inhibition and stimulation experiments showed that plasma paraoxonase 1 (PON1) is responsible for the hydrolysis of 2R3’R-SGM in humans and rats. The PON1-mediated hydrolysis of 2R3’R-SGM was confirmed in the lipoprotein-rich fractions of human plasma. As PON1 is naturally attached to high-density lipoprotein, it might be absent in topical tissues where 2R3’R-SGM is applied, supporting its local stability and efficacy. The metabolic behavior of 2R3’R-SGM indicates that it is an ideal soft drug to be detoxified as soon as it moves into systemic circulation. Furthermore, the similarity of 2R3’R-SGM metabolism in humans and rats showed that the rat is a suitable animal for preclinical study.     

Sublethal toxicity of esbiothrin relationship with total antioxidant status and in vivo genotoxicity assessment in fish (Cyprinus carpio L., 1758) using the micronucleus test and comet assay

Esbiothrin, synthetic pyrethroid with quick activity against insects, is widely used against household pests and in public health. Despite widespread use, data on ecotoxicity and genotoxic effects are extremely scarce. The aim of the present study is to evaluate the genotoxic potential of esbiothrin on a model fish species Cyprinus carpio L., 1758 (Pisces: Cyprinidae, koi) using the micronucleus test and comet assay in peripheral blood erythrocytes. Effects of two sublethal exposure concentrations on plasma total antioxidant status (TAS mmol/L), and Hct values were examined. On the basis of the 96 h LC₅₀ data from U.S. EPA ecotox database (32 μg/L) two sublethal exposure concentrations (5 and 10 μg/L) were used together with ethyl methanesulfonate (EMS) (5 mg/L) as positive control. Five fish were used for each dose/duration group (24, 48, and 72 h) under controlled laboratory conditions. The fish showed behavioral changes at the higher dose. Plasma TAS (mmol/L) levels decreased in 24 h; an increase was observed slightly for 48 and obviously for 72 h in both exposure doses. Similarly, hematocrit (Hct) values differed between exposure duration but no significant differences in mean values were found between groups of the same exposure time. The general trend was a rise after 48 h, which decreased afterwards. Our results revealed significant increases in the frequencies of micronuclei and levels of DNA strand breaks and thus demonstrated the genotoxic potential of this pesticide on fish, a nontarget organism of the aquatic ecosystem. To our knowledge this is the first study to report observable genotoxic effects of esbiothrin on fish. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28:644–651, 2013.     

Identification of regions of the P2X(7) receptor that contribute to human and rat species differences in antagonist effects

Background and purpose:

Several P2X₇ receptor antagonists are allosteric inhibitors and exhibit species difference in potency. Furthermore, N²-(3,4-difluorophenyl)-N¹-(2-methyl-5-(1-piperazinylmethyl)phenyl)glycinamide dihydrochloride ({"type":"entrez-nucleotide","attrs":{"text":"GW791343","term_id":"293587509","term_text":"GW791343"}}GW791343) exhibits negative allosteric effects at the human P2X₇ receptor but is a positive allosteric modulator of the rat P2X₇ receptor. In this study we have identified several regions of the P2X₇ receptor that contribute to the species differences in antagonist effects.

Experimental approach:

Chimeric human-rat P2X₇ receptors were constructed with regions of the rat receptor being inserted into the human receptor. Antagonist effects at these receptors were measured in ethidium accumulation and radioligand binding studies.

Key results:

Exchanging regions of the P2X₇ receptor close to transmembrane domain 1 modified the effects of KN62, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and {"type":"entrez-nucleotide","attrs":{"text":"GW791343","term_id":"293587509","term_text":"GW791343"}}GW791343. Further studies, in which single amino acids were exchanged, identified amino acid 95 as being primarily responsible for the differential allosteric effects of {"type":"entrez-nucleotide","attrs":{"text":"GW791343","term_id":"293587509","term_text":"GW791343"}}GW791343 and, to varying degrees, the species differences in potency of SB203580 and KN62. The species selectivity of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid was affected by multiple regions of the receptor, with potency being particularly affected by the amino acid 126 but not by amino acid 95. A further region of the rat receptor (amino acids 154–183) was identified that, when inserted into the corresponding position in the human receptor, increased ATP potency 10-fold.

Conclusions:

This study has identified several key residues responsible for the species differences in antagonist effects at the P2X₇ receptor and also identified a further region of the P2X₇ receptor that can significantly affect agonist potency at the P2X₇ receptor.     

DNA authentication of So-jutsu (Atractylodes lancea rhizome) and Byaku-jutsu (Atractylodes rhizome) obtained in the market based on the nucleotide sequence of the 18S–5.8S rDNA internal transcribed spacer region

The internal transcribed spacer 1 (ITS1) region between 18S and 5.8S rDNA was amplified from 25 crude drug specimens of So-jutsu (Atractylodes lancea rhizome) and 11 specimens of Byaku-jutsu (Atractylodes rhizome) obtained in the Chinese market. Based on the comparison of the nucleotide sequences of the PCR products with those determined in five Atractylodes species, we were able to identify the plant origins of these crude drugs. Of the 25 specimens of So-jutsu analyzed, 20 were identified to be derived from either A. chinensis, A. lancea or the interspecific hybrid between these two species, whereas five specimens were identified to be derived from A. japonica, i.e. Byaku-jutsu. In contrast, all of the specimens of Byaku-jutsu analyzed were derived from either A. ovata or A. japonica. A PCR-based method was established for the rapid identification and/or discrimination of So-jutsu and Byaku-jutsu without sequencing.     

Mode of action based risk assessment of the botanical food-borne alkenylbenzene apiol from parsley using physiologically based kinetic (PBK) modelling and read-across from safrole

The present study developed physiologically-based kinetic (PBK) models for the alkenylbenzene apiol in order to facilitate risk assessment based on read-across from the related alkenylbenzene safrole. Model predictions indicate that in rat liver the formation of the 1′-sulfoxy metabolite is about 3 times lower for apiol than for safrole. These data support that the lower confidence limit of the benchmark dose resulting in a 10% extra cancer incidence (BMDL₁₀) that would be obtained in a rodent carcinogenicity study with apiol may be 3-fold higher for apiol than for safrole. These results enable a preliminary risk assessment for apiol, for which tumor data are not available, using a BMDL₁₀ value of 3 times the BMDL₁₀ for safrole. Based on an estimated BMDL₁₀ for apiol of 5.7–15.3 mg/kg body wt per day and an estimated daily intake of 4 × 10⁻⁵ mg/kg body wt per day, the margin of exposure (MOE) would amount to 140,000–385,000. This indicates a low priority for risk management. The present study shows how PBK modelling can contribute to the development of alternatives for animal testing, facilitating read-across from compounds for which in vivo toxicity studies on tumor formation are available to compounds for which these data are unavailable.     

Reassessing the ichthyotoxin profile of cultured Prymnesium parvum (golden algae) and comparing it to samples collected from recent freshwater bloom and fish kill events in North America

Within the last two decades, Prymnesium parvum (golden algae) has rapidly spread into inland waterways across the southern portion of North America and this organism has now appeared in more northerly distributed watersheds. In its wake, golden algae blooms have left an alarming trail of ecological devastation, namely massive fish kills, which are threatening the economic and recreational value of freshwater systems throughout the United States. To further understand the nature of this emerging crisis, our group investigated the chemical nature of the toxin(s) produced by P. parvum. We approached the problem using a two-pronged strategy that included analyzing both laboratory-grown golden algae and field-collected samples of P. parvum. Our results demonstrate that there is a striking difference in the toxin profiles for these two systems. An assemblage of potently ichthyotoxic fatty acids consisting primarily of stearidonic acid was identified in P. parvum cultures. While the concentration of the fatty acids alone was sufficient to account for the rapid-onset ichthyotoxic properties of cultured P. parvum, we also detected a second type of highly labile ichthyotoxic substance(s) in laboratory-grown golden algae that remains uncharacterized. In contrast, the amounts of stearidonic acid and its related congeners present in samples from recent bloom and fish kill sites fell well below the limits necessary to induce acute toxicity in fish. However, a highly labile ichthyotoxic substance, which is similar to the one found in laboratory-grown P. parvum cultures, was also detected. We propose that the uncharacterized labile metabolite produced by P. parvum is responsible for golden algae's devastating fish killing effects. Moreover, we have determined that the biologically-relevant ichthyotoxins produced by P. parvum are not the prymnesins as is widely believed. Our results suggest that further intensive efforts will be required to chemically define P. parvum's ichthyotoxins under natural bloom conditions.     

The influence of fetal and neonatal exposure to dichlorodiphenyltrichloroethane (DDT) on the testosterone status of neonatal male rat

The testicular synthesis and hepatic metabolism of testosterone and the plasma concentrations of androgens were measured in 2-, 4-, 6-, 8-, or 10-day-old male rats after a 10-day prenatal and variable postnatal exposure to dichlorodiphenyltrichloroethane (DDT) via maternal milk secretion. While there were significant elevations in the metabolism of testosterone and aminopyrine by liver microsomes from DDT-exposed rats there were no compensatory changes in either serum androgens or testicular synthesis of testosterone. The failure of DDT exposure to change plasma concentrations of androgens in spite of substantial increase in hepatic metabolism was further confirmed in male rats treated with human chorionic gonadotrophin to produce elevated testosterone concentrations. Tissue analysis showed ¹⁴C-labeled DDT and/or its metabolites crossed the placenta and was present in fetal rats (Day 21), but cross fostering of litters from treated and control rats indicated that the major response to DDT occurs upon exposure after birth. It is suggested that hepatic enzyme induction by DDT is essentially a postnatal phenomenon and the DDT is transferred in substantial amounts from the milk of lactating rats to their offspring.     

On the biomarkers and mechanisms of konzo, a distinct upper motor neuron disease associated with food (cassava) cyanogenic exposure

Konzo is a self-limiting central motor-system disease associated with food dependency on cassava and low dietary intake of sulfur amino acids (SAA). Under conditions of SAA-deficiency, ingested cassava cyanogens yield metabolites that include thiocyanate and cyanate, a protein-carbamoylating agent. We studied the physical and biochemical modifications of rat serum and spinal cord proteins arising from intoxication of young adult rats with 50-200 mg/kg linamarin, or 200 mg/kg sodium cyanate (NaOCN), or vehicle (saline) and fed either a normal amino acid- or SAA-deficient diet for up to 2 weeks. Animals under SAA-deficient diet and treatment with linamarin or NaOCN developed hind limb tremors or motor weakness, respectively. LC/MS-MS analysis revealed differential albumin carbamoylation in animals treated with NaOCN, vs. linamarin/SAA-deficient diet, or vehicle. 2D-DIGE and MALDI-TOF/MS-MS analysis of the spinal cord proteome showed differential expression of proteins involved in oxidative mechanisms (e.g. peroxiredoxin 6), endocytic vesicular trafficking (e.g. dynamin 1), protein folding (e.g. protein disulfide isomerase), and maintenance of the cytoskeleton integrity (e.g. α-spectrin). Studies are needed to elucidate the role of the aformentioned modifications in the pathogenesis of cassava-associated motor-system disease.     

Translation,cross-cultural adaptation and validation of the revised Patients’ Attitudes Towards Deprescribing (rPATD) questionnaire to Portuguese: Older adults version

BackgroundDeprescribing is a patient-centered approach to managing inappropriate polypharmacy that faces several barriers, including patients' attitudes and beliefs about medications that need to be considered. For this purpose, the revised Patients' Attitudes Towards Deprescribing (rPATD) questionnaire is a helpful instrument, but until now, there is no Portuguese version.ObjectivesTo translate and validate the rPATD questionnaire (older adults version) to Portuguese.MethodsThe rPATD questionnaire was translated and cross-culturally adapted using forward-backward translation and pre-testing. A cross-sectional study with 192 older adults aged ≥65 years taking at least 1 regular medication was conducted for validity assessment. Participants were recruited by convenience sampling in 3 Portuguese outpatient rehabilitation medicine clinics. Psychometric properties were evaluated through face and content validity; construct validity, by assessing structural validity through exploratory factor analysis, hypotheses testing, including concurrent validity and cross cultural validity; reliability with internal consistency; and item-total correlation. Floor and ceiling effects were examined.ResultsThe exploratory factor analysis (EFA) revealed a 4-factor structure that explains 51.08% of the total variance, as in the original rPATD. These 4 factors are related to the level of involvement in medication management, beliefs in the appropriateness of medication, perceived burden of medication, and concerns about stopping medications. Factor loadings ranged from 0.226 to 0.800; 2 items scored <0.3, and no cross-loading was found. The exclusion of the 2 items loading <0.3 in the EFA showed no significant improvement in factor loading or internal consistency, so the item structure was maintained. In hypothesis testing, 78% of the correlations were correctly predicted. The 4 factors internal consistency was generally acceptable, with Cronbach's alpha ranging from 0.638 to 0.830. The item-total correlation ranged between 0.223 and 0.7.ConclusionThe Portuguese rPATD questionnaire for older adults presents globally good or acceptable psychometric properties.     

Effects of intravenous kainic acid,N-methyl-d-aspartate,and (-)-nuciferine on the cat spinal cord

Summary Kainic acid (a rigid conformational analogue of glutamate), N-methyl-d-aspartate (the methylated derivative of aspartate), and (-)-nuciferine (an aporphine alkaloid with a depressant effect on glutamate-induced neuronal firing), which, so far, have been examined in microiontophoretic studies, were investigated in spinal cats for their effects on some spinal cord activities after intravenous injections.At low doses, kainic acid (0.3 mg kg^–1) enhanced segmental monosynaptic but not polysynaptic ventral root reflexes and increased the excitability of motoneurones, whereas N-methyl-d-aspartate (3 mg kg^–1) facilitated polysynaptic but not monosynaptic reflexes. Higher doses of the two amino acids depolarized motoneurones and primary afferent endings, enhanced monosynaptic reflexes and depressed polysynaptic reflexes.(-)-Nuciferine (1–10 mg kg^–1) depressed monosynaptic but not polysynaptic ventral root reflexes in a dose-dependent manner and antagonized the effects of kainic acid but not of N-methyl-d-aspartate on the spinal cord.The results are consistent with the hypothetical excitatory transmitter role of glutamate in primary afferents and of aspartate in excitatory spinal cord interneurones; the findings also suggest that (-)-nuciferine may be used as a systemically effective, rather selective blocker of central glutamate receptors.     

Anti-oxidative and anti-inflammatory activities of some isolated constituents from the stem bark of Allanblackia monticola Staner L.C (Guttiferae)

Stem bark of Allanblackia monticola has been used in association with others plant in the Cameroonian folk medicine for the treatment of various diseases such amoebic dysentery, diarrhoea, lung infections, and skin diseases. The methylene chloride fraction, its isolated compounds like α-mangostin, lupeol and acid betulinic were screened for antioxidant activity using free radical scavenging method. These isolated compounds were further tested for anti-inflammatory properties using carrageenan-induced model. Methylene chloride fraction, showed concentration-dependent radical scavenging activity, by inhibiting 1,1-diphenyl-1-picryl-hydrazyl radical (DPPH) with an IC₅₀ value of 14.60 μg/ml. α-Mangostin and betulinic acid (500 μg/ml), showed weak radical scavenging activity with a maximum inhibition reaching 38.07 μg/ml and 26.38 μg/ml, respectively. Betulinic acid, lupeol and α-mangostin (5 mg/kg and 9.37 mg/kg) showed anti-inflammatory activity with a maximum inhibition of 57.89%, 57.14% and 38.70%, respectively. Methylene chloride fraction of Allanblackia monticola and some derivatives, have antioxidant and anti-inflammatory activities. Received 7 July 2008; accepted 7 October 2008     

Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay, the PP2A inhibition assay and HPLC with fluorimetric detection

An approach involving chemical, functional and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos and Saronikos Gulfs, Greece, during DSP episodes that occurred in 2006-2007. Samples were analyzed using the mouse bioassay, high performance liquid chromatography with fluorimetric detection (HPLC-FLD), using l-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, and the protein phosphatase 2A inhibition assay (PP2AIA) using a commercially available kit. Okadaic acid (OA) and its polar and non-polar esters were detected and quantified by HPLC-FLD, after hydrolysis of the samples during preparation. The detection limit of the HPLC method for OA was 5.86 μg OA/kg, which permits this method to be used for the regulatory control of these toxins in shellfish. Comparison of the results by all three methods revealed excellent consistency.     

Characterizing the effect of UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A9 genetic polymorphisms on enantioselective glucuronidation of flurbiprofen

Flurbiprofen (FPF), available commercially as a racemic mixture, is a propionic acid derivative of non-steroidal anti-inflammatory drugs (NSAIDs) with known stereoselective glucuronidation. The major enzyme catalyzing this conjugation reaction is UDP-glucuronosyltransferase (UGT) 2B7, with minor contributions by UGT1A9. This study examines the role of the genetic variants of UGT2B7 and 1A9 enzymes involved in the formation of acyl glucuronides (FPFGs). Variants caused by three single nucleotide polymorphisms (SNPs) (A71S, 211G>T; H268Y, 802C>T; and D398N, 1192G>A) in UGT2B7 and three SNPs (C3Y, 8G>A; M33T, 98T>C; D256N, 766G>A) in UGT1A9 showed activity changes toward different substrates. However the functional impacts of these SNPs on chiral substrates were not examined. Upon stable expression in Bac-to-Bac system, UGT2B7*71S (A⁷¹S), UGT2B7*2 (H²⁶⁸Y) and UGT2B7*5 (D³⁹⁸N) were all associated with a decrease in the formation of FPFGs. Compared with UGT2B7*1 (wild-type), UGT2B7*71S exhibited a >2-fold lower intrinsic clearance mainly by altered capacities (V_max). Furthermore, a >14-fold decreased intrinsic clearance of the *1 protein was produced by UGT2B7*2 and UGT2B7*5. However, no significantly stereoselective difference for the formation of (R)- and (S)-FPFG was found among these UGT2B7 allozymes. UGT1A9*2 (C³Y) exhibited a higher V_max (3.2-fold), K_m (2.1-fold) and intrinsic clearance (1.6-fold) toward (S)-FPF than UGT1A9*1 (wild-type). UGT1A9*3 (M³³T) almost lost the catalytic activity to FPF. A significantly stereoselective difference on the glucuronidation of rac-FPF was seen between the two variants compared with the wild type of UGT1A9.     

Alterations in protein metabolism and amino acid concentrations in rats fed by a high-protein (casein-enriched) diet – Effect of starvation

Rats were fed with a standard laboratory diet (SLD) or a high-protein diet (HPD). After three months changes in amino acid concentration and protein metabolism were examined in fed and 24 h-fasted animals. In the blood of the HPD animals sacrificed in fed state were found higher concentrations of urea, aspartate, taurine, proline, valine, isoleucine, and leucine, and lower concentrations of glycine and cysteine. The main alterations in tissues were decreased concentrations of glycine and increased concentrations of valine, isoleucine, and leucine. Differences in weight, protein concentration, protein synthesis, and proteolysis in tissues were insignificant. The exception was soleus muscle in which higher values of protein synthesis and proteolysis were found in HPD animals. The response to starvation of HPD and SLD fed animals was different. In animals fed before starvation by HPD was found more pronounced decrease in a number of individual amino acids in plasma and tissues and more pronounced decrease in protein synthesis in muscle, spleen, jejunum, and colon. It is concluded that chronic intake of HPD has not positive effect on protein balance in any tissue, results in the imbalance in aminoacidemia in extracellular and intracellular fluid, and alters the response of the organism to starvation.     

Agonist binding and activation of the rat beta(1)-adrenergic receptor: role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43))

We investigated the role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43)) in agonist binding to, and activation of, the rat beta(1)-adrenergic receptor by comparing pK(i)s and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD(2) and pK(i) (P > 0.4), suggesting that changes in pK(i) were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr(356(7.43)). For the other agonists, the change in pK(i) values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat beta(1)-adrenergic receptor, Trp(134(3.28)) and Tyr(356(7.43)) form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp(134(3.28)) and Tyr(356(7.43)), together with Trp(353(7.40)), are able to interact via pi-pi interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser(190(4.57)) appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.