Relationship between α-glycerophosphate dehydrogenase activity and metabolic rate during flight in Drosophila melanogaster

Measurements of wing-beat frequency (WBF) have been used to characterize flight muscle metabolic rate in Drosophila melanogaster during tethered flight. Progeny of crosses between 17 X-chromosome substitution lines and three null-activity stocks have been studied in order to determine the effect on flight metabolism of sharply reduced activity of -glycerophosphate dehydrogenase (GPDH). It was found that flies with an approximate 50% reduction in GPDH activity have a metabolic rate that is, in most cases, indistinguishable from that of wild-type flies and, in the most extreme cases, reduced by only 4%. These results demonstrate that Gpdh is not a major gene for flight metabolism, in the quantitative genetic sense of the term. These results are in agreement with the Kacser and Burns (1973, 1979, 1981) theory of flux, which postulates that the activity of an enzyme embedded in a multienzyme pathway can sometimes vary from wild-type to very low levels (perhaps 5–10% wild type) with no significant effect on flux through the total pathway.This research was supported by several grants to JWC: NSF Grant 8211667, a grant from the Graduate School, University of Minnesota, and a Research Career Development Award from the NIH.     

Synthesis ofα-triazolylα-amino acid derivatives

Summary We report the synthesis of-triazolyl-amino esters by 1,3 dipolar cycloaddition of acetylenic compounds and-azido-amino esters.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Mictic patterns of the rotifer Brachionus plicatilis Müller in small ponds

Populations of the rotifer Brachionus plicatilis were monitored in three small ponds in a marsh on the Mediterranean coast. Samples were taken approximately every three weeks from July 1992 to November 1993. Salinity, temperature, conductivity, pH and oxygen concentration were measured in the field. Population density was determined from preserved quantitative samples. Individuals were classified as mictic females, amictic females, non-ovigerous females, and males, differentiating between two morphotypes (S and L). From these counts, a level of mixis was calculated. We also determined the proportion of mictic females in natural populations by culturing females isolated from fresh samples. From these data, mictic patterns over time and correlation between levels of mixis and environmental and population parameters were analyzed. From a previous study S and L morphotypes were known to correspond to genetically different clonal groups. Our data showed that reproduction was predominantly parthenogenetic in these clonal groups, but mictic females were found in most samples, the proportion of mictic females ranging from 0 to 29%. The clonal groups showed different patterns of mixis. L clonal group presented a continuous sexual reproductive pattern. In contrast, S clones showed a rather punctuated mictic pattern. A positive correlation between levels of sexual reproduction and population density was found for S and L groups. However, they differed in their density threshold for mictic reproduction. The adaptive meaning of these patterns and their implications in maintaining genetic diversity within and between populations are discussed.     

Identification of a locality in snake venomα-neurotoxins with a singificant compositional similarity to marine snailα-conotoxins: Implications for evolution and structure/activity

Summary -neurotoxins from elapid snake venoms and-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an-neurotoxin from the false sea snakeLaticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller-conotoxins (from the marine snailsConus magus andConus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake-neurotoxins and their homologues. Six Proteins unrelated to erabutoxin b were compared to the-conotoxins to show that the extent of the erabutoxin b/-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely-cobratoxin fromNaja naja siamensis and cytotoxin V^II4 fromNaja mossambica mossambica, were also analyzed. The extent of the matching with the-conotoxins decreased in the series erabutoxin b>-cobratoxin>cytotoxin V^II4, and this also relates the order of similarity to the pharmacological properties of the-conotoxins.The-conotoxin-like area of the snake-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.     

Genetic control of chalcone-flavanone isomerase activity in Callistephus chinensis

A mutant blocked in anthocyanin synthesis leads to an accumulation of 4,2,4,6-tetrahydroxy-chalcone-2-glucoside (isosalipurposide) in blossoms of Callistephus chinesis (L.) Nees, whereas in geno-types with the wild-type allele, higher oxidized flavonoids and anthocyanins are synthesized. Measurements of chalcone-flavanone isomerase activity of 18 lines of Callistephus chinensis showed a clear correlation between accumulation of chalcone in the recessive genotypes (ch ch) and deficiency of this enzyme activity. Both the chemogenetic and the enzymologic evidence lead to the following conclusions: 1. The first product of the synthesis of the flavonoid skeleton should be tetrahydroxychalcone.-2. The chalcone-flavanone isomerase catalyzes the formation of flavanone from chalcone in a stereospecific way and there-with furnishes the substrate for the further reactions in the flavonoid biosynthesis.Abbreviations EGME ethylene glycol monomethyl ether - HOAc acetic acid - MeOH methanol - PVP polyvinylpyrrolidone - TBA tert. butanol-acetic acid-water, 3:1:1 - TLC thin-layer chromatography     

Linkage mapping of ‘25-kDa globulin’ genes on homoeologous group-1 chromosomes of bread and durum wheat

Acid polyacrylamide-gel electrophoresis (A-PAGE) of ethanol-soluble proteins from the endosperm of bread and durum wheats reveals some bands encoded by genes on the homoeologous group-1 chromosomes with higher mobility than the -gliadins. The isolation of these proteins showed that they were the previously described 25-kDa globulins encoded by genes at the Glo-A1, Glo-B1, and Glo-D1 loci. The variability found among a collection of 51 bread and 81 durum wheats was very low: two allelic variants at Glo-A1 and no variants at Glo-B1 in each of the two species, and two allelic variants at Glo-D1 in bread wheat. Inheritance studies of 25-kDa globulin genes on group-1 chromosomes of bread and durum wheat were carried out on the F₂ progeny from four crosses, two in bread wheat and two in durum wheat. The linkage mapping of the 1A 25-kDa globulin genes of bread wheat was done based on four prolamin loci: Glu-A1, Glu-A3, Gli-A1 and Gli -A3. The percentages of recombination and the distances found allowed a re-evaluation of the linkage map of endosperm protein loci on this chromosome. The Glo-A1 locus was found to be located at the distal end of the short arm of 1A chromosome, at a distance of 5.23±1.99 cM from Gli-A1, 6.85±2.22 cM from Glu-A3, 22.64±3.62 cM from Gli-A1, and at a recombination percentage of 49.30±4.40 from Glu-A1. A similar distance between Gli-A1 and Glo-A1 (4.82±1.75 and 6.66±2.26 cM) was found in durum wheat. The distance between Gli-D1 and Glo-D1 on chromosome 1D was 2.86±1.39 cM.     

Classification ofPythium ‘group F’ based on mycelial protein and isozyme patterns

Taxonomic characteristics were compared among 10 isolates ofPythium group F in tems of the electrophoretic patterns of their mycelial proteins and isozymes. These isolates were obtained from water of three ponds in different seasons and have an identical morphology of zoosporangia. Attempts to cross the isolates with each other themselves and with other isolates from the same group failed.Pythium group F is the most dominant of the pythia in the aquatic ecosystem and is difficult to identify because of the lack of sexual reproductive structures. Isozyme analysis proved useful in this respect. Comparisons of banding patterns of total soluble proteins and isozymes revealed five subgroups inPythium group F. Two isolates obtained from water of different ponds in different seasons showed the same protein and isozyme patterns. Our findings indicate that the use of total soluble protein and isozyme patterns for determining the variation withinPythium group F could become a valuable adjunct to the morphological and physiological criteria.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

Tremorgenic mycotoxins produced by strains of Penicillium spp. isolated from toxic Poa huecu parodi

Seventeen strains of Penicillium spp. have been isolated from Poa huecu Parodi from the Zapala zone, exhibiting toxicity to sheet. The following strains have been identified: P. crustosum, cyclopium, notatum, palitans, puberulum, verrucosum, viridicatum and Penicillium spp. The toxigenic capacity of the strains was studied after growing them under suitable conditions. Toxins produced were analysed by thin layer chromatography (TLC). Penitrem A (PA) and Penitrem B (PB) neurotoxins were identified and quantitated in twelve strains; verruculogen (VERR) and fumitremorgen B (FTB) being present in one of them. The effect of these mycotoxins was studied in mice. Neurological symptoms characteristic of the intoxication by tremorgenic toxins and similar to those observed in sheep suffering from huecu's disease were observed. The possible role of these toxins as causative agents of huecu's disease is discussed.     

DNA polymorphisms in the ψβ1- and β-globin gene regions in asian macaques

DNA polymorphisms in the ₁--globin gene region in nine Asian macaques(Macaca fuscata, M. mulatta, M. nemestrina, M. cyclopis, M. fascicularis, M. arctoides, M. radiata, M. maura, andM. assamensis) were examined using several restriction endonucleases and the human ₁, IVS2, and IVS2 probes. TheBamHI site 3 to the -globin gene was polymorphic inM. fuscata andM. mulatta, while the HincII site and the EcoRI site in the ₁-globin gene region was highly polymorphic inM. fuscata andM. mulatta, respectively. These polymorphic sites also seem to be present in other Asian macaques. The present study of the polymorphism at theBamHI site 3 to the -globin gene in Asian macaques supports, at the nuclear DNA level, the idea that thefascicularis group includingM. fuscata, M. mulatta, M. cyclopis, andM. fascicularis is different from other Asian macaque groups.This study was supported in part by the Cooperation Research Program of the Primate Research Institute, Kyoto University.     

Evidence suggesting energy-dependent formaldehyde transport in an RuMP-type methylotroph (T15)

Formaldehyde accumulation ratios ([¹⁴CH₂O]_i/[¹⁴CH₂O]_o) as high as 12-fold were measured in anaerobic, CH₃OH-energized, whole cell suspensions of the ribulose monophosphate (RuMP)-type methylotrophic strain T15. Uptake kinetics were extremely rapid, enabling the attainment of equilibrium in only 10–30 s. Transport appears to be energy-dependent and associated with the protonmotive force (pmf). Anaerobic incubation with 5 M carbonyl p-(trifluoromethoxy)-phenylhydrazone (FCCP) led to 70%–90% reduction of the accumulation ratio. Though not as pronounced, diminished uptake was also observed in the presence of 140 M nigericin, 161 M valinomycin and 90 mM KSCN, commensurate with their effects on pmf. Accumulation of CH₂O as a function of external pH followed a trend more similar to that of pmf than either pH or . Preventing energization by incubation with 100 M N,N-dicyclohexylcarbodiimide (DCCD) led to nearly 80% inhibition of CH₂O transport. Over short time periods it was possible to chase accumulated ¹⁴CH₂O from previously loaded cells by collapsing pmf; however, this technique also indicated that significant ¹⁴CH₂O incorporation began to occur within 3 min.Abbreviations FCCP Carbonyl cyanide p-(trifluoromethyoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - RuMP ribulose monophosphate - TPP⁺ tetra[U-¹⁴C]phenylphosphonium - pmf protonmotive force     

Prediction of the secondary structure content of globular proteins based on structural classes

The prediction of the secondary structure content (-helix and-strand content) of a globular protein may play an important complementary role in the prediction of the protein's structure. We propose a new prediction algorithm based on Chou's database [Chou (1995),Proteins Struct. Fund Genet. 21, 319]. The new algorithm is an improved multiple linear regression method, taking the nonlinear and coupling terms of the frequencies of different amino acids into account. The prediction is also based on the structural classes of proteins. A resubstitution examination for the algorithm shows that the average errors are 0.040 and 0.033 for the prediction of-helix content and-strand content, respectively. The examination of cross-validation, the jackknife analysis, shows that the average errors are 0.051 and 0.044 for the prediction of-helix content and-strand content, respectively. Both examinations indicate the self-consistency and the extrapolative effectiveness of the new algorithm. Compared with the other methods available currently, our method has the merits of simplicity and convenience for use, as well as a high prediction accuracy. By incorporating the prediction of the structural classes, the only input of our method is the amino acid composition of the protein to be predicted.