Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects Are Enzyme and Substrate Dependent

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K _m, increasing its V _max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K _m are concerned. In the cases of V _max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V _max increases. Additionally, the BSA effects may be UGT subfamily dependent since K _m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V _max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.     

Cloning, sequencing, and characterization of the iturin A operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.     

A, B, and H isoantigens in the human fetus

We studied the appearance of isoantigens A, B, and H during the fertilization age in human embryos and fetuses with the specific red cell adherence (SRCA) test. The first positive reaction was found in endothelial cells of a 4-wk-old embryo. In 6-wk-old embryos the isoantigens appeared on the luminal surface of the epithelium of the stomach and small intestine. In other organs the blood group isoantigens were detected at different stages of development; in 12-wk-old fetuses the distribution became identical with that of adults. In pancreatic exocrine glands from fetuses of all blood groups up to 5 mo old, H antigen alone was present. Brunner's glands of the duodenum reacted positively for both A and B blood group antigens of the fetus and also for H antigen, which supports the assumption that some secretory glands produce the precursor H substance throughout fetal and adult life.     

Motherhood,Apple Pie,Hemoglobin A1c,and the DCCT

ObjectiveTo review recent literature on the limitations of hemoglobin A_1c (HbA_1c) as a marker of glycemic control.MethodsEnglish-language literature published between 1985 and 2011 was reviewed specific to analyses of major trials relating glycemic control to complications of diabetes mellitus, as expressed through HbA_1c as a marker of glycemic control.ResultsHbA_1c has been accepted as the most fundamental biomarker in diabetes, if not all of medicine, as it clearly predicts risk for diabetes-related complications. What is not generally appreciated is that HbA_1c is a crude marker of glycemia with many limitations. It is now accepted that HbA_1c does not reflect mean glucose for many people, and even for those it does, any level could represent a wide range of glycemia. While we have learned HbA_1c is not a perfect biomarker, we also know that in the Diabetes Control and Complications Trial, HbA_1c could only explain 11% of the variation in retinopathy risk between the conventional and intensive therapy groups. This important finding suggests that other glycemic and nonglycemic factors may be responsible for the pathogenesis of diabetes-related complications. One candidate is glycemic variability, which must be differentiated from postprandial hyperglycemia since hypoglycemia can also result in inflammatory activation. Importantly, although it is clear that in insulin-requiring patients glycemic variability is associated with hypoglycemia, we require a definitive prospective trial to confirm glycemic variability’s association with one or more vascular complications.ConclusionsWhat is abundantly clear is that the HbA_1c message, as we know it, is too simplistic. While certain wholesome concepts such as motherhood and apple pie are accepted by all, the HbA_1c message may be more complex than originally appreciated, and it may be time to reevaluate our most basic premise in diabetes. (Endocr Pract. 2012;18:78-84)     

R A Fisher,design theory,and the Indian connection

Design Theory, a branch of mathematics, was born out of the experimental statistics research of the population geneticist R A Fisher and of Indian mathematical statisticians in the 1930s. The field combines elements of combinatorics, finite projective geometries, Latin squares, and a variety of further mathematical structures, brought together in surprising ways. This essay will present these structures and ideas as well as how the field came together, in itself an interesting story.     

A New Harziane Diterpene,Harziaketal A,and a New Sterol,Trichosterol A,from the Marine-Alga-Epiphytic Trichoderma sp. Z43

One new diterpene, harziaketal A ( 1 ), and one new highly degraded sterol, trichosterol A ( 2 ), along with three known compounds, including one diterpene, harzianone ( 3 ), and two steroids, (22E,24R)-5α,6α-epoxy-ergosta-8(14),22-dien-3β,7α-diol ( 4 ) and isoergokonin B ( 5 ), were isolated from the culture of the marine-alga-epiphytic fungus Trichoderma sp. Z43 by silica gel column chromatography (CC), Sephadex LH-20 CC, and preparative thin-layer chromatography (TLC). Their structures and relative configurations were assigned by nuclear magnetic resonance (NMR) and high resolution electrospray ionisation mass spectrometry (HR-ESI-MS) data, and the absolute configuration of 1 was established by X-ray diffraction. Compound 1 features a hemiketal unit situated at the four-membered ring of harziane-type diterpenes for the first time, while 2 represents the rare occurrence of sterols with rings A and B being degraded. Compounds 1 and 2 displayed weak inhibition against the tested phytoplankton (Amphidinium carterae, Heterocapsa circularisquama, Heterosigma akashiwo, and Prorocentrum donghaiense) with half maximal inhibitory concentration (IC₅₀) ranging from 14 to 53 μg/mL.     

Interactions of the Growth Retardants Daminozide and Piproctanyl Bromide, and Gibberellins A1, A3, A4+7, A5 and A13 in Stem Extension and Inflorescence Development in Chrysanthemum morifolium Ramat

Interactions between the growth retardants daminozide (a substitutedsuccinamic acid) or piproctanyl bromide (a quaternary ammonium,piperidinium compound), and a subsequent application of a singledose (40 µg) of either gibberellin A₁, A₃, A₄₊₇ or A₁₂,showed that, in Chrysanthemum morifolium Ramat. cv. Bright GoldenAnne, a strong interdependence exists between elongation ofthe lateral shoot and the rate of development of its terminalinflorescence. A₁, A₃, and A₄₊₇ were highly active in overcoming the restrictionson both internode extension and the rate of flower-bud developmentimposed by either retardant, suggesting that these two retardanteffects are caused by a deficiency of active gibberellins (GN).In the absence of retardant, A₁, A₃, and A₄₊₇ markedly increasedstem elongation, and flowering occurred earlier than in plantsreceiving neither retardant nor GN. A₁₃ the only 20-carbon GNtested, was much less active, while A₅ had a relatively greatereffect on the time of flowering than on shoot elongation. Thus,it is not necessarily the rate of stem extension which determinesthe rate of inflorescence development. The response to different amounts of A₁, A₃ or A₁₃ (1, 5, 10,20, or 50 µg per shoot) neither suggest that different‘threshold’ levels of a particular GN are requiredto induce increases in either stem elongation or in the rateat which inflorescences develop, nor did a change in the dosegiven lead to any consistent differential effect on these twoprocesses. Chrysanthemum morifolium Ramat., stem extension, inflorescence development, growth retardants, gibberellins     

Multiplex PCR for the discrimination of A. fumigatus, A. flavus, A. niger and A. terreus

Aspergillus pathogens usually infect immunocompromised patients with lethal outcome. We report a multiplex PCR assay for the discrimination of the most frequent Aspergillus pathogens, A. fumigatus, A. flavus, A. niger and A. terreus, through distinct amplicons of 250 bp, 200 bp, 150 bp and 450 bp respectively, derived from the rDNA gene of A. terreus and the aspergillopepsin genes of the remaining species.     

Homoeology and phylogeny of the A,S, and D genomes of the triticinae

Summary Two-dimensional gel electrophoresis of seedling proteins has been undertaken on diploid representatives of the A, S, and DTriticum genomes. The 457 reproducible spots of the hexaploid Chinese Spring were taken into account in the comparisons. More than 82% of the hexaploid spots were found among the representatives of each of the three genomes. Thus, it was proposed that the homoeologous structural loci, on the three homoeologous genomes, can be present in the same allelic forms and that from 72% to 86% of the spots of the hexaploid pattern are in two or three doses. The dendrogram, deduced from the similarity indices computed between the 16 diploid accessions, was very consistent with our present knowledge of the genetic relationships in theTriticum genus. This result, in addition to previous ones, shows that the comparison of two-dimensional protein patterns represents a novel experimental approach for studying the phylogenetic relationships in theTriticinae.     

A comparative cytogenetic study of the tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi

Differential C-banding of chromosomes and in situ hybridization with the probes pTa71 and pTa794 were used for a comparative cytogenetic study of the three tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi. These species were similar in the structure and patterns of C-banding of several chromosomes as well as in the location of the loci 5S rRNA genes and main NOR sites; however, they differed in the number and localization of minor 45S rDNA loci as well as in the morphology and distribution of heterochromatin in some chromosomes. According to the data obtained, A. insularis is closer to A. magna, whereas A. murphyi is somewhat separated from these two species. Presumably, all the three studied species originated from the same tetraploid ancestor, and their divergence is connected with various species-specific chromosome rearrangements. The evolution of A. murphyi is likely to have occurred independently of the other two species.     

Reassessment of the evolution of wheat chromosomes 4A, 5A,and 7B

Key message

Comparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B.

Abstract

Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements.

     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.