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.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

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.     

Immunohistochemical localisation of substances reactive to antisera against α-and β-endorphin and met-enkephalin in the brain of Rana temporaria L.

Summary In the brain of Rana temporaria, two distinct systems reactive with - and -endorphin antisera, respectively, and with a met-enkephalin antiserum, have been detected immunohistochemically.Neurons reacting with - and -endorphin antisera are located (1) in the preoptic nucleus, and (2) in the pars ventralis of the tuber cinereum. Immunoreactive nerve fibres of both groups of perikarya end in the infundibular floor near the capillaries and the preoptico-hypophysial tract. Control reactions have shown that the immunoreactivity is suppressed by the corresponding antigens, but also by -LPH. In view of these results the immunoreactive systems examined correspond to an /-endorphin system or a lipotropinergic system.Neurons reacting with the met-enkephalin antiserum are located in the paraventricular organ. Intense immunofluorescence was observed in the infundibular floor. Controls show that the labelling by met-enkephalin antiserum is exclusively suppressed by met-enkephalin.In the pituitary gland, on the other hand, - and -endorphin antisera reveal: 1) the MSH/ACTH-like cells of the pars intermedia and 2) the ACTH-like cells of the pars distalis.Supported by the D.G.R.S.T., Contrat no 77.7.0648     

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins

Primary structure analysis of the four river buffalo -globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two -globin chains, ^I1 and ^II3, which differ at positions 129 and 131: ^I1 has 64 Ala, 129 Phe, 131 Asn; ^II3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two -globin chains, ^I2 and ^II4, which differ at positions 10 and 11: ^I2 has 10 Ile, 11 Gln, 64 Asn; ^II4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic -globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo -globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.     

β-Glucosidase excretion by Trichoderma pseudokoningii: Correlation with cell wall bound β-1.3-glucanase activities

The formation and excretion of -glucosidase from Trichoderma pseudokoningii was studied during growth on different carbon sources. The enzyme was present under all conditions examined, but increased activity was found during growth on carbon sources favouring slow growth.Two different patterns of -glucosidase excretion were observed: on carbon sources allowing fast growth a relatively high percentage of total activity was found in the culture fluid, which decreases as the culture grows older, but which increases again during the phase of cell lysis; on carbon sources favouring slow growth, excretion is initially low, but commences at later culture stages.Changes in cell wall composition and cell wall lytic enzyme activities associated with the cell walls were examined during phases of high and low ratios of extracellular to cell-wall bound -glucosidase activities. With no component of the cell wall (chitin, -glucan, -glucan, galactosamine) could correlation with -glucosidase excretion be identified. Among a number of cell-wall lytic, cell-wall associated enzymes (-glucanases, -glucanases, glucosaminidase, galactosaminidase), -1.3-glucanase activity correlated well with the excretion of -glucosidase.The results suggest a possible role of -1.3-glucanases in the mechanism of release of -glucosidase from cell walls of T. pseudokoningii; this is discussed.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Laser kinetic spectroscopy studies of the bimolecular oxygenation of alpha- and beta-subunits within the R-state of human hemoglobin

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants k = 27.2 ± 1.3 (M·sec)^-1 and k = 62.9 ± 1.6 (M·sec)^-1. Both the observed processes have been assigned to the bimolecular oxygenation of - and -subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are = 0.0120 ± 0.0017 and = 0.044 ± 0.005 for - and -subunits, respectively. The oxygenation reactions of isolated ^PCMB- and ^PCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants k = 44 ± 2 (M·sec)^-1 and k = 51 ± 1 (M·sec)^-1, respectively. The quantum yields of photodissociation of isolated ^PCMB- and ^PCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.     

Immunocytochemical localization of α-melanocyte-stimulating hormone in the brain of the lizard,Lacerta muralis

Summary The distribution of -melanocyte-stimulating hormone (-MSH) was studied in the brain of the lizard Lacerta muralis by means of immunocytochemical staining methods. -MSH-like containing cells were found in the ventro-lateral preoptic area and the paraventricular and supraoptic nuclei. Some scattered cells staining for -MSH were also detected in the mesencephalo-diencephalic boundary region, while numerous -MSH-like nerve fibres were localized in the medial eminence. No reaction was observed after the use of antiserum preabsorbed with synthetic antigen.These findings suggest that an -MSH-like peptidergic system could possibly be involved in the hypothalamo-hypophysial regulation and/or play a role as neurotransmitter in this animal.     

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy

Hypoxia has been shown to activate the pleiotropic cytokine TNF in the lung. TNF in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNF in chronic hypoxia we exposed TNF null (TNF–/–) and wild-type mice to three weeks of hypobaric hypoxia (10% O₂). Equivalent erythocytosis (Hematocrit increased by 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice ( 75%), yet was unaltered in the TNF–/– mice. Concomitantly right ventricular hypertrophy was attenuated in the TNF–/– mice (35 ± 5% increase) when compared to wild-type mice (124 ± 6% increase p < 0.001, n 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNF is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNF signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNF.     

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4–12-fold) stimulation of the activity of membrane-associated -glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsutum L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of -glucan synthesis. Those products whose synthesis is stimulated are mainly -1,3-glucan, but there is also a considerable increase in -1,4-glucan. No -1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on -glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.Abbreviations UDP-glucose uridine-5-diphosphoglucose - PEG polyethylene glycol - BTP bistrispropane (1,3-bis[tris(hydroxymethyl)methylamino]propane) - MES 2(N-morpholino)ethanesulfonic acid - VAL valinomycin     

Beyond translation: elongation factor-1α and the cytoskeleton

Summary While reported interactions of elongation factor-1 (EF-1) with various other molecules involved in protein biosynthesis are abundant, its interactions with major cytoskeletal proteins have not been as extensively examined. Major roles for EF-1 in cytoskeletal organization emerge from a review of such interactions within species as diverse as slime molds and mammals, sea urchins and higher plants. Based on these studies, the integration of EF-1's cytoskeletal roles with those of translation is considered, and prospective mechanisms for regulation of EF-1's cytoskeletal associations are discussed.Abbreviations EF elongation factor - RNP ribonucleoprotein particle - MT microtubule - MA mitotic apparatus - CaM calmodulin - MAP microtubule-associated protein     

Uptake and metabolism of α-aminoadipic acid by Penicillium chrysogenum wis 54-1255

The uptake of 1-¹⁴C-dl--aminoadipate in resting mycelium of Penicillium chrysogenum Wis 54-1255 and its metabolism during benzylpenicillin formation were studied. The pH optimum for uptake at 25°C was 6.4. Over a range of concentrations from 0.01–1.0 mM, approximately 45% of 1-¹⁴C-dl--aminoadipate was taken up by carbon-starved mycelium. ¹⁴CO₂ was formed at a low rate, and the total formed amounted to only 1–3% of the 1-¹⁴C-dl--aminoadipate supplied. The intracellular pool of -aminoadipate appears to be expandable, depending on the concentration of -aminoadipate in the medium. The rate of penicillin synthesis depended on the intracellular concentration of -aminoadipate. Penicillin biosynthesis achieved half of the maximum rate at an intracellular concentration of 0.06 nmol -aminoadipate/mg dry cell weight. This low concentration, the result of adding 0.01 mM dl--aminoadipate to the medium, was sufficient to reverse the inhibition of penicillin biosynthesis caused by 10 mM extracellular l-lysine. Aminoadipate appears to be recycled during penicillin formation. Labeled -ketoadipate was formed from -aminoadipate to the extent of about 25%.Abbreviation DCW dry cell weight     

α-Isopropylmalate synthase as a marker for the leucine biosynthetic pathway in several clostridia and in Bacteroides fragilis

-Isopropylmalate synthase (EC 4.1.3.12) is present in extracts of Bacteroides fragilis, Clostridium thermoaceticum, Clostridium formicoacetium, Clostridium pasteurianum, and Clostridium kluyveri with specific activities (mol -isopropylmalate formed per min and g protein) of 8.6, 8.9, 2.4, 1.9, and 0.3, respectively. The product -isopropylmalate was identified by gas chromatography combined with mass spectroscopy. The presence of 5 mM leucine in the growth medium represses the synthesis of -isopropylmalate synthase in C. thermoaceticum by 40 and 70 %. The enzyme from C. pasteurianum was partially purified to a specific activity of 1413. All studied enzyme properties are similar to those of the enzymes from aerobic bacteria. It is suggested that in these anaerobic bacteria the -isopropylmalate pathway is present in addition to the pathway via the ferrodoxin-dependent, reductive carboxylation of branched chain fatty acids.Abbreviations used -KIV -Ketoisovalerate - -IPM -Isopropylmalate - CoA Coenzyme A     

A model for cleavage ofO-glycosidic bonds in glycoproteins

The present work investigated the possibility of cleavage of -linkages between mannose or galactose and serine/threonine residues by -mannosidase and -galactosidase. The study was carried out initially with model synthetic compounds imitating theO-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that -mannosidase and -galactosidase can hydrolyse these linkages after proteolytic digestion of glucosamylase.     

Labdane diterpene derivatives from Holwaya mucida

Clumps of white crystals present in 40-day-old malt agar cultures of Holwaya mucida were isolated as long white needles by crystallization from ethanol following short extraction with chloroform. The levorotary compound ([] ₂₈₉ ²¹ =-193.8°) was recognized as a -lactone (C₁₇H₂₀O₅) by infrared and mass spectrometry. It was identified as 7-methoxy-3a, 10b-dimethyl-1, 2, 3, 3a, 5a, 7, 10b, 10c-octahydro-4H, 9H-furo[2, 3, 4 : 4, 5]naphtho[2, 1-c]pyran-4, 9-dione, a labdane-derived compound known as antibiotic LL-Z1271. Preparative thin-layer chromatography of the mother liquor afforded 2 minor metabolites. One was identified as LL-Z1271, the demethylated analogue of LL-Z1271. The other one named LL-Z1271, was recognized as a compound related to and : its structure could not be fully elucidated. H. mucida (anamorph: Crinula calciiformis) has no taxonomic relationship with two other LL-Z1271 producing species viz. Acrostalagmus sp. (= Acremonium cf. atrogriseum) and Oidiodendron truncatum.     

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