α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Cajaflavanone and cajanone released from Cajanus cajan (L. Millsp.) roots induce nod genes of Bradyrhizobium sp.

A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium sp (F-4) nod DABC-lacZ translation fusion was constructed and used to monitor nod gene expression in response to pigeonpea root exudate. Two nod-inducing compounds were isolated and identified. Spectral analysis using ultraviolet absorption, infrared spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 5,4-dihydroxy-6-(3-methyl-2-butenyl)-2, 2-dimethyl pyrano-[5, 6:7, 8]-flavanone (cajaflavanone) and 2,4,5-trihydroxy-5-isopentenyl-6, 7-dimethylchromene iso-flavanone (cajanone). When pEA2-21 was introduced into Rhizobium trifolii and R. meliloti cajanone and cajaflavanone did not induce nod gene indicating that specificity of induction appears to be influenced by the host-strain genome.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Characterization and gene organization of Taiwan banded krait (Bungarus multicinctus) gamma-bungarotoxin

-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with -sheet structure as is that of snake venom -neurotoxins and cardiotoxins. -Bungarotoxin exhibits activity on inhibiting the binding of [³H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated -bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC₅₀ of approximately 200 nM. The genomic DNA encoding the -bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for -neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that -bungarotoxin, -neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.     

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.     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

Mating reaction in Saccharomyces cerevisiae

A diffusible sex-specific substance called substance-I (S-I) was isolated from culture filtrate of type strains of the yeast Saccharomyces cerevisiae. The isolated S-I, an oligopeptide, induced sexual cell agglutinability in inducible a type strains and enhanced the agglutinability in constitutive a type strains. The induction of sexual agglutinability was detected in 30 min and reached maximum in 90 min, when 0.2 g/ml of S-I was added to inducible a type cells. The a type-specific factor responsible for sexual cell agglutination, called a type agglutination factor (aAF), was shown to be produced during the induction or the enhancement of agglutinability of a type cells by S-I. The aAF produced in response to S-I was not different in the susceptibility to proteolytic enzymes and disulfide-cleaving agents from those produced constitutively in the absence of S-I.     

Ovarian development in Culex nigripalpus and its implication for disease transmission

Ovarian development was investigated in wild and colonized Culex nigripalpus Theobald females. Blood-seeking females with morphologically distinct nulliparous, parous and resorbed ovarian follicles (as opposed to females with degenerating follicles) were collected from the field all year, indicating year around gonoactivity. During the cooler months a larger number of females with resorbed follicles were collected from the field than in warmer months. Laboratory experiments showed that resorbed ovarian follicles occurred in sucrose fed females when deprived of a blood meal after emergence. These resorbed ovarian follicles were functional, since most of them began to develop and mature after the females took a blood meal to repletion. Among females fed a partial blood meal, some did not begin ovarian development and, therefore, exhibited gonotrophic dissociation. Females fed partial blood meals had similar patterns of ovarian development, irrespective of temperature at which they were maintained. Implications of occurrence of females with resorbed ovarian follicles and disease transmission are discussed.Supported in part by National Institutes of Health Research Grants No. A1-06587 and RR-07021. A contribution of the Florida Medical Entomology Laboratory, Vero Beach, Florida, Florida Agriculture Experimental Station Journal Series No. 2367     

Aırborne pollen calendar of Bartın, Turkey

Our study comprises airborne pollen analysis of the years 1995–1997 of Bartn (40°38 N–32°21 E). The aims of the study were: to determine the pollen types present in the atmosphere, to calculate their actual concentrations, and to establish the pollen calendar for Bartn. For this purpose, pollen slides of Durham apparatus, which was located in four different locations in Bartn, were examined on a weekly basis. In this study, 19062 pollen grains were counted and 18484 of those were determined on the slides in the air of Bartn during three years of period. Results show that 13758 of 18484 pollen grains belong to arboreal, 4726 belong to non-arboreal taxa. 3698 of determined non-arboreal taxa belong to Gramineae.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

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     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

α-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an -lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of -lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, -lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and -ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an -lipoic acid, coenzyme A, and pyruvate or -ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an -lipoic acid (K _0.5=1.4±0.8 mM) and lipoamide (K _0.5=0.9±0.3 mM) dependent manner.     

New organisms—novel genetics

This report describes a method for quantifying -endotoxin contents in spray-dried preparations ofBacillus thuringiensis strain GC-91. -Endotoxin is solubilized in the pro-toxin form and separated from other soluble compounds by ion exchange chromatography. The method does not discriminate between the different -endotoxins produced by strain GC-91. It is suitable for quality control, since -endotoxin concentrations found in different preparations correlate inversely with the LC 50 in bioassays on artificial diet against freshly hatched larvae ofSpodoptera littoralis. A typical batch of spray-dried material contains 1.22%±0.08% -endotoxin. The method is most accurate with preparations containing 0.5 to 2.0% toxin, for which triplicate estimations give standard errors close to±0.2%.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose