Synthesis of aminobenzyltriethylenetetraaminohexaacetic acid: conjugation of the chelator to protein by an alkylamine linkage

The conjugation of a chelating agent to an antibody as an anchoring site for a radionuclide is the first step in the successful preparation of a radiolabeled antibody for a diagnostic and therapeutic application. The high affinity of the protein bound chelator towards radionuclide ensures a higher selectivity in the delivery of the radionuclide to the targeted tissue. 4-Aminobenzylderivativetriethlenetetraaminohexaacetic acid (TTHA), a hexadentate chelating agent has been now prepared for conjugation with proteins in view of the higher affinity of TTHA metal ions as compared to DTPA. The latent crosslinking potential of -hydroxy aldehydes has been used to conjugate the new chelating agent to proteins through an alkylamine linkage. On incubation of amino benzyl TTHA with glycoladehyde at neutral pH and room temperature, the reagent is converted to oxoethyl amino benzyl TTHA. On addition of albumin to this reaction mixture, the oxo ethylamino benzyl TTHA generates reversible schiff base adducts with the amino groups of albumin. The reduction of the Schiff base adducts of the chelator with the protein by sodium cyanoborohydride stabilizes the schiff base adducts as stable alkylamine linkages. 4-Thiocyanatobenzyl TTHA has also been prepared and conjugated to albumin through a thiocarbamoyl linkage. Both preparations of TTHA conjugated albumin complexed with ^99mTc and ¹¹¹In, with high affinity and no decomposition of the complex was noticed for at least up to 6 hrs after the preparation. The radiolabels complexed with these TTHA -albumin conjugates could not be chased out by free DTPA. A comparison of the biodistribution of ¹¹¹In, bound to the TTHA conjugated through an alkylamine and a thiocarbamoyl linkage showed that ¹¹¹In complexed with alkylamine linked TTHA was retained in blood to a level nearly 17% higher compared to that seen with thicarbamoyl linked TTHA, one hr after the injection into mice. Thus, the alkylamine linkage appears to be more stable under the in vivo conditions. The glycolaldehyde mediated alkylation procedure offers a mild, simple and rapid method for preparation of drug-protein (antibody) conjugates.     

The state of the environment of the Loosdrecht lakes

The Loosdrecht lakes are a system of shallow, interconnected, peat lakes in the centre of The Netherlands. The main environmental functions of the Loosdrecht lakes are nature and recreation. From the point of view of the Dutch policy, a Specific Environmental Quality (Bijzondere Milieukwaliteit) should be set for these lakes.The most serious environmental problem of the area is eutrophication. The Loosdrecht lakes have, by increasing external phosphorus loading, changed, from clear lakes with few macrophytes, followed by a period of abundant characean growth, to turbid lakes dominated by cyanobacteria and detrital matter. Eutrophication was counteracted by use of sewerage systems and dephosporization of the supply water. The resultant decrease in external phosphorus loading did not result in a decrease of turbidity by suspended particles.The eutrophication of the lake ecosystems was described as a series of phases. One of those phases, the status around 1940, has been used as an ecological reference system.By means of a graphical presentation technique, the so-called AMOEBE-approach, the state of the environment of the Loosdrecht lakes has been visualized. Thirty-two ecological parameters, including both biotic and abiotic factors, have been selected and quantified. Concrete target values for these parameters have been derived from historical reports and from Lake Western Loenderveen, located close to the Loosdrecht lakes, but less eutrophic.The general conclusion is that the state of the environment of the Loosdrecht lakes is far from what is required with respect to a Specific Environmental Quality, as many of the selected parameters, like water transparency, total phosphorus, mineral nitrogen, cyanobacteria, bream, pike, macrophytes, birds and otter, deviate by over an order of magnitude from their desired levels.     

Stable isotopic composition of otoliths from tagged Pacific halibut, Hippoglossus stenolepis

Data from tagging studies and stable isotope ratio analyses (¹⁸O and ¹³C) in otoliths of tagged Pacific halibut, Hippoglossus stenolepis, provided unique information about halibut distribution and area migration. The ¹⁸O values of otoliths of individual halibut tagged in the Bering Sea and the Gulf of Alaska ranged from –0.5 to +1.0 VPDB (Vienna Peedee belemnite) in the first few years, but gradually increased to 2.0–2.5 VPDB when these tagged fish were recaptured as adults in waters off British Columbia. Similar isotopic variations were found in ¹³C. Comparison of isotopic composition from the age-1 halibut (i.e., aragonite samples taken from the first annulus of otoliths) and the adult (ages 8) indicated that the ocean conditions between the Bering Sea and the northeast Pacific were markedly different over the tagging period, but the area boundaries between the Gulf of Alaska and British Columbia were not clear. We concluded that stable isotopic records in otoliths of tagged Pacific halibut were informative and particularly useful for details of subsequent movement of halibut between release and recovery.     

Seasonal Stable Isotope Records of Otoliths from Ocean-pen Reared and Wild Cod, Gadus morhua

Otoliths of ocean-pen reared cod, Gadus morhua, provide a unique opportunity to examine the lifetime history of the fish. Here we report 18 analyses of such otoliths on seasonal (winter and summer) stable oxygen and carbon isotope ratios. The calculated isotopic temperatures from otoliths of reared cod were essentially in agreement with the temperature record during rearing, suggesting that temperature is a dominant factor in the precipitation of otolith aragonite. Carbon isotope ratios increased with age and leveled off at 4 years old, presumed to correlate with sexual maturity of cod. As compared with otoliths of wild-caught cod, significant differences were found in isotope variation and the correlation between ¹³C and ¹⁸O. These differences were probably attributable to the different environmental constraints and food supply.     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Effect of 2-guanidinoethanol on levels of monoamines and their metabolites in the rat brain

The contents of monoamines and their metabolites in rat brains 3 hours after the intracerebroventricular injection of 6 mol of 2-guanidino-ethanol (GEt) were measured by HPLC. GEt which is a configurational analogue of 4-aminobutanoic acid (GABA) induced severe running fits and tonic-clonic convulsions as well as epileptic discharges. In GEt-administered rats, dopamine (DA) decreased in the cortex, hippocampus and hypothalamus. 3,4-Dihydroxyphenylacetic acid (DOPAC) increased to about the same level in all brain regions, therefore the distribution of DOPAC appeared to be homogeneous in the brain. The homovanillic acid levels also increased in the striatum and hippocampus. No significant change in the norepinephrine contents was observed in any region. The turnover ratio of DA increased significantly except in the striatum. Serotonin levels increased in the hypothalamus and midbrain by GEt administration, though 5-hydroxyindoleacetic acid levels showed no change in any of the brain regions. These data suggest that the activity of dopaminergic and serotonergic neurons are increased by GEt.     

An efficient synthesis of 3′-amino-3′-deoxythymidine derivatives

An efficient method of reduction of 3-azido-3-deoxythymidine and its 5-protected derivatives to 3-aminothymidine derivatives on a palladium catalyst using ammonium formate as a source of hydrogen was suggested.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 147–150.Original Russian Text Copyright © 2005 by Seregin, Chudinov, Yurkevich, Shvets.     

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans

The -amylase gene (amy) from Streptomyces griseus IMRU 3570 and the -galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P₂ or P₃. The -amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum. Correspondence to: J. F. Martín     

CD studies of peptides that mimic the four helicoidal sequences of the uteroglobin monomer

Summary Short peptides spanning the helicoidal sequences of the uteroglobin monomer (crystal forms P2₁ and C222₁) were synthesized and studied by circular dichroism spectroscopy. None of them showed any secondary structure in the absence of HFIP. However, most peptides achieved a helical conformation when this structuring agent was used, with the exception of the analogue corresponding to the helicoidal fragment 19–24 (helix II, crystal P2₁). These results indicate that other factors, such as interchain interactions, have to contribute to helix stabilization in the molecule. On the other hand, while peptides corresponding to N- and C-terminal fragments that contain the first and fourth helices of the monomer, respectively (1–14 and 48–70) achieved a -like structure when 10–15% of HFIP was used, this behaviour was not observed when TFE was used. Moreover, substitution of cysteine by -aminobutyric acid at position 3 increased both the helicity of fragment 1–14 and its ability to adopt a -like structure, but the opposite effect was observed for fragment 48–70 when -aminobutyric acid was introduced at position 69. These results indicate that this part of the protein might be sensitive to the chemical environment it is exposed to and that the two cysteine residues at positions 3 and 69 of the monomer could play a different role in the folding process.     

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas

When programmed with yeast prepro--factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp--F_cyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp--F₃, 27.5 kDa). Glycosylation of the membrane specific pp--F₃ species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp--F₀) , whereas the primary translation product pp--Fcyt is not affected. Likewise, only the glycosylated pp--F₃ structure is digested by Endo H yielding a polypeptide with a molecular mass between PP--F₀ and pp--Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp--Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp--F₃ polypeptide indicates that its oligosaccharide chains are processed to presumbly Man₉-GlcNAc₂ structures under thein vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence. In addition, several arguments suggest that both trimming enzymes are located in the lumen of the microsomal vesicles derived from endoplasmic reticulum membranes.Abbreviations dNM 1-deoxynojirimycin - N-Me-dNM N-methyl-dNM - dMM 1-deoxymannojirimycin - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Mutagen sensitivity and mutability in lentil

Summary Seeds of two cultivars, each of macrosperma and microsperma varietal groups of lentil were mutagenised with gamma-rays and NMU to determine their mutagen sensitivity and mutability. The increasing doeses of gamma-rays and NMU decreased germination, root and shoot length, pollen fertility and plant survival, but increased the occurrence of leaf spots. The root system was found to be more sensitive to both mutagens than the shoot. The macrosperma varietal group was more sensitive to both the mutagens than microsperma group. In microsperma group, variety Pusa-1 was more sensitive to both the mutagens than L-259, while in the macrosperma group L-1491 showed more sensitivity to the mutagens than L-1492. Radio-sensitivity corresponded positively with chemosensitivity in both varietal groups. There was a positive relationship between radio- and chemo-sensitivity of the genotypes and their mutability. The results also revealed the existence of a parallelism between radiomutability and chemo-mutability. Due to saturation in the mutational events and vigour of both diplontic and haplontic selection in the biological material, the mutation frequency either decreased or remained constant at higher doses of the mutagens.     

Selective cytotoxicity of scFv-TNF fusion protein on the tumor cells

A fusion protein of single-chain, Fv-tumor necrosis factor , scFv-TNF, was constructed and expressed in the Pichia pastoris expression system. There was 67-fold less toxicity of the fusion protein when compared with TNF- alone in cells expressing CA125. Furthermore, scFv-TNF was 10-fold more selective in cell killing directed by anti-CA125 scFv in a CA125-positive cell line than the CA125-negative line.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

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     

Biochemical basis of winter wheat resistance to the grain aphid, Sitobion avenae

Six winter wheat cultivars with differing degrees of resistance to the grain aphid, Sitobion avenae (F.), were studied under field conditions. Resistance was measured in terms of non-preference and antibiosis on plants at seven growth stages. The varieties Saga and Grana were most resistant in terms of both non-preference and antibiosis to S. avenae at all growth stages examined. The varieties Liwilla and Dana were relatively susceptible to aphid attack. The number of aphids was directly proportional to the total content of free and essential amino acids. The level of resistance showed a similar, but not identical, relationship with the observed concentration of soluble proteins. A higher degree of cultivar resistance was associated with a higher value on a toxicity index, which is the ratio between the free-phenols and free-amino acids content. Obtained results suggest that the resistance of winter wheat cultivars to the grain aphid was based mainly on the mechanism of antibiosis.

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Résumé Six cultivars de blé d'hiver, présentant différents degrés de résistance au puceron des grains, S. avenae, ont été étudiés dans la nature. La résistance a été évaluée en fonction de l'antibiose et de l'absence de préférence de plantes à 7 stades de développement. Les variétés Saga et Grana ont été les plus résistantes, suivant ces deux critères, pour tous les stades de puceron étudiés. Les variétés Liwilla et Dana ont été relativement sensibles. Les effectifs de pucerons étaient proportionnels à la teneur totale en acides aminés libres essentiels. Les concentrations en protéines solubles présentent une relation de même type, mais pas identique, avec le niveau de résistance. Un haut degré de résistance a été associé à une valeur élevée de l'indice de toxicité, rapport entre les phénols libres et la teneur en acides aminés libres. Ces résultats suggèrent une résistance des cultivars de blé d'hiver à S. avenae liée principalement aux mécanismes d'antibiose.

     

Salt-induced hypertension in WKY rats: Prevention by α-lipoic acid supplementation

There is strong evidence that points to excess dietary salt as a major factor contributing to the development of hypertension. Salt sensitivity is associated with glucose intolerance and insulin resistance in both animal models and humans. In insulin resistance, impaired glucose metabolism leads to elevated endogenous aldehydes which bind to vascular calcium channels, increasing cytosolic [Ca²⁺]_i and blood pressure. In an insulin resistant animal model of hypertension, spontaneously hypertensive rats (SHRs), dietary supplementation with lipoic acid lowers tissue aldehydes and plasma insulin levels and normalizes blood pressure. The objective of this study is to examine the effects of a high salt diet on tissue aldehydes, cytosolic [Ca²⁺]_i and blood pressure in WKY rats and to investigate whether dietary supplementation with lipoic acid can prevent a salt induced increase in blood pressure. Starting at 7 weeks of age, WKY rats were divided into three groups of six animals each and treated for 10 weeks with diets as follows: WKY-normal salt (0.7% NaCl); WKY-high salt (8% NaCl); WKY-high salt + lipoic acid (8% NaCl diet + lipoic acid 500 mg/Kg feed). At completion, animals in the high salt group had elevated systolic blood pressure, platelet [Ca²⁺]_i, and tissue aldehyde conjugates compared with the normal salt group and showed smooth muscle cell hyperplasia in the small arteries and arterioles of the kidneys. Dietary -lipoic acid supplementation in high salt-treated WKY rats normalized systolic blood pressure and cytosolic [Ca²⁺]_i and aldehydes in liver and aorta. Kidney aldehydes and renal vascular changes were attenuated, but not normalized.