bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of the skin mitogens tumor‐promotor 12‐O‐tetradecanoylphorbol 13‐acetate and divalent‐cation‐ionophore A23187 on ion fluxes and membrane potential in a murine epidermal cell line (HEL30) and in 3T3 fibroblasts
Authors: SEEMANN Dietmar , FÜRSTENBERGER Gerhard , MARKS Friedrich
Published: March 2005

The transmembrane potential of HEL30 keratinocytes and 3T3 fibroblasts has been determined by measuring the distribution of labelled triphenylmethylphosphonium bromide. The tumor‐promotor 12‐O‐tetradecanoylphorbol 13‐acetate (1–5μM) induces hyperpolarization in 3T3 cells but does not exert any effect on the membrane potential of keratinocytes, whereas the divalent cation ionophore A23187 (0.5–1 μM) hyperpolarizes keratinocytes and probably also 3T3 cells.

Studies on Na+ and Rb+ fluxes, as well as with different inhibitors, indicate that the hyperpolarizing effect is the consequence of an increased Na+ influx which in turn stimulates the Na+/K+‐dependent ATPase. No causal relationship seems to exist between the change of the membrane potential and arachidonic acid release (and subsequent prostaglandin synthesis) which is induced by both drugs in both cell lines.

Since the induction of the arachidonic cascade (by both agents) as well as the stimulation of Na+ influx (by A23187) are found to be critically dependent on extracellular Ca2+ and are inhibited by ‘Ca2+‐blockers’, it is concluded that both reactions are triggered by the same event (Ca2+ translocation) but proceed independently of each other. The release of arachidonic acid is already stimulated under conditions where a measurable influx of Ca2+ is not yet observed. This indicates a local mobilization of Ca2+, perhaps across the plasma membrane.

It is concluded that monovalent cation fluxes and changes of the membrane potential are not critically involved in the stimulation of the arachidonic acid cascade and cellular proliferation by agents which induce epidermal hyperplasia in vivo.
Conformational properties of phospholipases A2
Authors: DUFTON Mark J. , EAKER David , HIDER Robert C.
Published: March 2005

The sequences of 32 phospholipases A2 (EC 3.1.1.4) were analysed by secondary‐structure prediction and the results were compared with the available crystallographic data. Good agreement is evident between prediction and experiment, especially for helical structure. Circular dichroic spectra were also determined for six enzymes from Elapid snake venom and these, in association with previously published spectra, confirm the main implication of the predictions, namely that all the homologues have qualitatively similar tertiary structures.

Consideration was then given to possible structure/activity relationships in the light of the above findings. The relative hydrophobicity/hydrophilicity of the area of the enzyme thought to interact with lipid/water interfaces was predicted and certain correlations were noted with relative penetrating power, species of origin and the presence of β‐neurotoxic properties.
Isolation and characterization of a cytochrome b–f complex from pea chloroplasts
Authors: PHILLIPS Andrew L. , GRAY John C.
Published: March 2005

A cytochrome b–f complex has been extracted from pea chloroplast from pea chloroplast membranes with octyl glucoside and cholate and has been purified by ammonium sulphate fractionation and polyacrylamide gel electrophoresis in the presence of Triton X‐100 and sodium deoxycholate. The complex contains cytochromes f and b‐563 in a ratio of 1:2, but shows very low levels of the Rieske iron sulphur centre and plastoquinol‐plastocyanin oxidoreductase activity. The complex consists of four polypeptides of molecular weights 37300, 34000, 19500 and 15200. The complex is dissociated by electrophoresis in the presence of 2M urea to give pure preparations of cytochrome f and cytochrome b‐563, which on sodium dodecylsulphate‐polyacrylamide gel electrophoresis give single bands of apparent molecular weights 37300 and 19500 respectively.
Changes in proteoglycan composition of F9 teratocarcinoma cells upon differentiation
Authors: KAPOOR Rajesh , PREHM Peter
Published: March 2005

Teratocarcinoma cells (F9) were induced to differentiate by retinoic acid and then labelled with [3H]‐glucosamine and [35S]‐sulphate. Proteoglycans were then isolated from the plasma membranes and the culture medium by mild, dissociative, non‐shear‐dependent techniques. The undifferentiated cells were devoid of hyaluronic acid and contained negligible quantities of heparan sulphate, dermatan sulphate and chondroitin sulphate. Upon differentiation, the cells synthesized large amounts of hyaluronic acid and there was a threefold increase in the amount of membrane‐bound sulphated proteoglycans. The differentiated cells also synthesized a proteoglycan (PGM‐2) which was shed completely into the medium. It consisted of a large protein core with covalently linked sugar chains which were sulphated and had an approximate molecular weight of 12000. These sugar chains consisted of glucosamine and galactose in a molar ratio of 1:1 and contained a small quantity of mannose. Upon differentiation of the cells the amount of this molecule increased by threefold. This molecule was distinct from other proteoglycans since it was resistant to degradation by heparanase, chondroitinases, hyaluronidase and neuraminidase, but could be degraded by keratanase. Structurally it was very similar to keratan sulphate, consisting of alternating residues of (‐Gal‐GlcNAc‐) in chains of approximately 20 such disaccharide units.
The expression of different monooxygenases supported by cytochrome P‐450 in neonatal rats and in primary fetal hepatocytes in culture
Authors: KREMERS Pierre , LETAWE‐GOUJON Francine , DE GRAEVE Jean , DUVIVIER Joseph , GIELEN Jacques E. , BASTIN Michèle , FRANKINET‐COLLIGNON Colette , WOLFF Dany
Published: March 2005

In rat liver, the perinatal development of various monooxygenase activities follows different patterns, depending upon the reaction studied. The ontogeny of the 6β‐, 7α‐ and 16α‐testosterone hydroxylase activities differs very significantly.

Aldrin epoxidase and steroid‐metabolizing monooxygenases are expressed in primary fetal rat liver cells in culture after treatment in vitro with dexamethasone. Testosterone is not metabolized by the control cells and is hydroxylated on the 6β and 16α positions following the addition of corticoids to the culture medium. The dose and time curves vary according to the hydroxylated position of the steroid. Aldrin epoxidase activity is nearly undetectable in the control cells, but is present and is inducible by phenobarbital following treatment with the corticoid. Phenobarbital induces aldrin epoxidase in the absence of dexamethasone in the culture medium, providing that the cells are pretreated with the corticoid for 48 h. The use of antibodies against the main cytochrome P‐450 species purified from adult and phenobarbital‐treated rats confirms that a similar cytochrome P‐450 can be induced in fetal cells in culture.

The perinatal regulation of biological events, such as the expression of the monooxygenases, can be reproduced in fetal rat liver cells in culture; such a model constitutes a unique tool for studying the biochemical mechanisms which control these phenomena.
Biogenesis of blowfly photoreceptor membranes is regulated by 11‐cis‐retinal
Authors: PAULSEN Reinhard , SCHWEMER Joachim
Published: March 2005

Biogenesis of photoreceptor membranes has been investigated by analyzing the rhodopsin and opsin content of microvillar photoreceptor membranes after injecting retinal isomers and radioactive amino acids into the compound eyes of carotenoid‐deficient blowflies. The amount of rhodopsin in the membranes was measured photometrically in extracts of isolated rhabdoms. The opsin content of the membranes and the level of radioactive labelling of opsin were measured after separating the membrane proteins by dodecyl sulphate/polyacrylamide gel electrophoresis.

1. In the photoreceptor membrane of carotenoid‐deficient flies the rhodopsin and opsin content is less than 4% of that in normal flies where opsin may constitute about 65% of the total membrane protein.

2. In carotenoid‐deficient flies the incorporation of rhodopsin into photoreceptor membranes is triggered by 11‐cis‐retinal but not all‐trans retinal. After injection of 11‐cis‐retinal the opsin content of the photoreceptor membranes increases in parallel with the rhodopsin content.

3. Radioactive labelling of opsin reveals that 11‐cis‐retinal triggers an incorporation of newly synthesized opsin into photoreceptor membranes whereas all‐trans retinal does not induce the assembly of opsin into photoreceptor membranes. Light‐dependent incorporation of radioactive labelled opsin into membranes with a high rhodopsin content shows that not only the visual pigment chromophore but also the opsin undergoes a light‐dependent turnover. The findings raise the possibility that opsin synthesis in blowfly photoreceptors and consequently the assembly of photoreceptor membranes is regulated by 11‐cis‐retinal.
The insulin‐stimulated receptor kinase is a tyrosine‐specific casein kinase
Authors: ZICK Yehiel , REES‐JONES Robert W. , GRUNBERGER George , TAYLOR Simeon I. , MONCADA Victoria , GORDEN Phillip , ROTH Jesse
Published: March 2005

Insulin stimulates a kinase that phosphorylates tyrosines in the insulin receptor; this kinase is tightly associated with the insulin receptor itself. We now show that the insulin‐stimulated casein kinase, present in solubilized, lectin‐purified receptor preparations from rat liver, is indistinguishable from the insulin receptor kinase. As with phosphorylation of the insulin receptor, insulin selectively enhanced by 2–3‐fold the phosphorylation of tyrosines in casein. The insulin‐stimulated activities of both kinases were inactivated at 37°C with the same t0.5 of 5 min and were identically affected by alkylating agents. Both receptor and casein kinase activities were specifically coprecipitated by anti‐receptor antibodies or by insulin and anti‐insulin antibodies. When the latter type of immune complexes were incubated with an excess of insulin, both kinase activities were quantitatively recovered. We therefore conclude that insulin‐stimulated receptor and casein phosphorylations are probably catalyzed by a single enzyme which is tightly associated with the receptor itself. Now, by replacing casein for receptor as substrate, it is possible to measure the enzymatic activity of this receptor‐related kinase itself, i.e. independent of the receptor as substrate. Detection of this activity is improved in the presence of certain alkylating agents. Use of artificial substrates (in combination with alkylating agents) is particularly important to dissect the functional components of the receptor complex, to study mechanisms of enzyme regulation and especially in situations where the available receptor for study is limited, e.g. fresh or cultured cells from patients.
Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl‐tRNA synthetase
Authors: GARRET Maurice , LABOUESSE Bernard , LITVAK Simon , ROMBY Pascale , EBEL Jean Pierre , GIEGÉ Richard
Published: March 2005

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N‐7 in guanosines and N‐3 in cytidines by dimethyl sulfate and carbethoxylation of N‐7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl‐tRNA synthetase.

The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and TΨ arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable TΨ stem.

When alkylation by ethylnitrosourea was studied with the tRNATrp· tryptophanyl‐tRNA synthetase complex we found that phosphates located at the 5′ side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N‐3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase · tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl‐tRNA synthetase was confirmed in nuclease S1 mapping experiments.
Investigations on stimulation of lac transcription in vivo in Escherichia coli by cAMP analogues
Authors: SCHOLÜBBERS Hans‐Gerd , van KNIPPENBERG Peter H. , BARANIAK Janina , STEC Wojciech J. , MORR Michael , JASTORFF Bernd
Published: March 2005

The ability of 24 systematically modified analogues of adenosine 3′,5′‐monophosphate (cAMP) to enhance the synthesis of β‐galactosidase in glucose‐repressed Escherichia coli strains KNBL 1001 and cpd– Crookes has been investigated. The properties of the analogues in comparison with cAMP are, with only two exceptions, alike in both strains. Two analogues, 7‐deazaadenosine 3′,5′‐monophosphate (i.e. tubercidin 3′,5′‐monophosphate) and (Rp)‐adenosine 3′,5′‐monothionophosphate, exhibit higher biological activity than cAMP. The latter analogue is 50‐fold more active in both strains. Three analogues showed activities comparable to cAMP, four analogues were less active and 12 analogues were unable to antagonize catabolite repression. Structure‐activity correlations showed that the 2′OH‐, 3′O‐, 5′O‐, the negative charge and the 6‐amino group cannot be modified without losing biological activity in vivo, while the N‐1 and N‐7 in adenine are not essential. The interaction with the catabolite gene activator protein is stereoselective for an unmodified axial exocyclic oxygen. The results are compared to those obtained with cAMP analogues in E. coli in vitro and those obtained with the same analogues in protein‐kinase systems and Dictyostelium species. The model of McKay et al. [McKay, D. B., Weber, J. T. and Steitz, T. A. (1982) J. Biol. Chem. 257, 9518–9524] proposed for distinct chemical interactions of cAMP with the catabolite gene activator protein is discussed and supplemented by additional hydrogen bond interactions.
A rapidly light‐induced chloroplast protein with a high turnover coded for by pea nuclear DNA
Authors: MEYER Gabriele , KLOPPSTECH Klaus
Published: March 2005

Among the translation products obtained in vitro with mRNAs isolated from etiolated grown pea, or after different times of illumination following the etiolation, a 24000‐Mr protein has been observed in the very early phase of greening; its occurrence culminates at 2–4 h after the start of illumination.

From these data it is concluded that the corresponding mRNA appears in and disappears from the poly(A)‐containing RNA population within hours. The protein product has been characterized as the precursor for a 17000‐Mr, chloroplast protein; by means of post‐translational transport in vitro, the processed product becomes bound to chloroplast membranes.

A product of the same size can also be labeled in vivo with a maximum of incorporation of label at 6–8 h after illumination. This product decays with a half‐life of about 5 h.

These findings imply a regulatory function of the 17000‐Mr protein during the process of greening, possibly by synchronization of nuclear and chloroplast genomes. Other possibilities are considered.
Isolation and complete amino‐acid sequence of the small polypeptide from light‐harvesting pigment‐protein complex I (B870) of Rhodopseudomonas capsulata
Authors: TADROS Monier H. , SUTER Franz , SEYDEWITZ Hans H. , WITT Irene , ZUBER Herbert , DREWS Gerhart
Published: March 2005

The small bacteriochlorophyll‐binding polypeptide of the light‐harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N‐terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C‐terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.
Lipid A fractions analyzed by a technique involving thin‐layer chromatography and enzyme‐linked immunosorbent assay
Authors: MATTSBY‐BALTZER Inger , ALVING Carl R.
Published: March 2005

A modified enzyme‐linked immunosorbent assay (ELISA), with alkaline phosphatase as enzyme, was used for the study of antigenicity of lipid A fractions directly on thin‐layer chromatographic (TLC) plates. For visualization a gel slab containing the enzyme substrate was placed on the plate containing enzyme‐conjugated antibodies. The plate was read by a thin‐layer chromatogram spectrophotometer. The immunoassay was both highly specific and quite sensitive. Sensitivity was superior to levels obtained by staining the plate with molybdenum blue (for phosphate) or orcinol (for carbohydrate). Fractions of lipid A from Escherichia coli 0111, Shigella flexneri or Salmonella minnesota R595, after being separated by thin‐layer chromatography, were analyzed using rabbit anti‐(lipid A) serum. Patterns obtained by scanning the same plates for phosphate staining and for the TLC‐ELISA corresponded well. For comparison with TLC‐ELISA, an inhibition assay was run using a tube ELISA. The tube ELISA, run in aqueous medium, showed that fractions 6–8 (those having the highest RF values) had the least activities. In contrast, TLC‐ELISA did not detect large differences between fractions 2–7. This discrepancy probably reflected limited aqueous solubility of fractions 6 and 7. We conclude that TLC‐ELISA might reveal antigenic activities of lipids that could be missed by other methods. The data suggested that all fractions, except for fraction 8, were similar in their antigenicity by TLC‐ELISA. Differences in antigenicity between the fractions occurred when the fractions were tested in free form in an aqueous environment and these differences possibly could have been due to different solubilities of individual fractions.
Hydrophobic interactions affect hydrogen bond strengths in complexes between peptides and vancomycin or ristocetin
Authors: WILLIAMSON Michael P. , WILLIAMS Dudley H.
Published: March 2005

A study on complexes of the glycopeptide antibiotics vancomycin and ristocetin with various dipeptides and tripeptides shows that the intermolecular hydrogen bond strengths of the complexes are reasonably well correlated with their free energy of formation. Such correlation is not anticipated on the basis of a purely hydrophobic interaction of the peptide side‐chains with the antibiotics. It is also shown that the free energy changes observed are very different from those expected as a result of hydrophobic forces. These facts suggest that addition of a hydrophobic group not only allows hydrophobic bonding but also strengthens existing hydrogen bonds. The increased hydrogen bond strength can be an important factor in determining the overall binding energy.
The action of lanthanum ions and formaldehyde on the proton‐pumping function of bacteriorhodopsin
Authors: DRACHEV Alexander L. , DRACHEV Lel A. , KAULEN Andrey D. , KHITRINA Lyubov V.
Published: March 2005

The photochemical cycle and the proton‐pumping function of bacteriorhodopsin modified with lanthanum and formaldehyde has been studied. In both preparations, the M412 → BR 570 transition time has been found to increase considerably. The deceleration of the photochemical cycle has been shown to be accompanied by inhibition of the millisecond phase of the photoelectrical response of bacteriorhodopsin membranes associated with phospholipid‐impregnated collodion film. Photoelectrogenic activity measured with permeable ion probe in proteoliposomes was also inhibited. Effects of lanthanum were reversed by EDTA. Formation of M412 was slightly accelerated and the microsecond electrogenic phase was not affected by lanthanum and by formaldehyde. It is concluded that lanthanum, but not formaldehyde, can be used as a specific reversible inhibitor of the second half of the bacteriorhodopsin photocycle and of the associated H+ uptake on the cytoplasmic side of the halobacterial membrane. Possible mechanisms of these effects are discussed.
The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus
Authors: KOCH H. Uwe , HAAS Renate , FISCHER Werner
Published: March 2005

Pulse‐chase experiments with [2‐3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol → (glycerophospho)25‐diglucosyldiacylglycerol + 25 diacylglycerol.

Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor.

Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative.

In contrast to normal growth, energy deprivation caused an immediate switch‐over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.
The T‐region of Ti plasmids codes for an enzyme synthesizing indole‐3‐acetic acid
Authors: SCHRÖDER Gudrun , WAFFENSCHMIDT Sabine , WEILER Elmar W. , SCHRÖDER Joachim
Published: March 2005

Gene 2 from the T region of Ti plasmids appears to be expressed both in eucaryotic and in procaryotic systems. In transformed plant cells it participates in auxin‐controlled growth and differentiation, and in bacteria it is expressed into a defined protein of Mr 49000. We investigated the possibility that it codes for an enzyme involved in auxin biosynthesis.

Only extracts from Escherichia coli cells expressing gene 2 hydrolyzed indole‐3‐acetamide into a substance which was unambiguously identified as indole‐3‐acetic acid. The same reaction was found in Agrobacteria containing gene 2, but not in strains lacking the gene. Extracts from tobacco crown gall cells, but not from non‐transformed cells, showed the same enzyme activity, and the reaction product was also identified as indole‐3‐acetic acid. The results indicate that gene 2 of the T region, which participates in tumorous growth of plant cells, codes both in bacteria and in plants for an amidohydrolase involved in the biosynthesis of the plant hormone indole‐3‐acetic acid.
The influence of the internal content of negatively charged liposomes on their interaction with high‐density lipoprotein
Authors: VIDAL Michel , BIENVENUE Alain , SAINTE‐MARIE Josette , PHILIPPOT Jean
Published: March 2005

The release of the internal content of negatively charged phosphatidylcholine/phosphatidylserine vesicles under the influence of high density lipoprotein was studied. Under standard conditions (the same composition outside and inside the compartment) the leakage of negative liposomes increased significantly. However, a high internal concentration of calcein provoked a sealing effect, exhibited both in sucrose and in calcein release. This sealing effect is not related to the size of vesicles, the fluidity of the membrane, the distribution of phosphatidylserine molecules, or the membrane potential. Our data indicate that surface potential influences this effect, probably in addition to a lateral pressure effect such as with cholesterol. The surface potential, as measured by the water‐lipid partition coefficient of fatty acids, is strongly affected by internal ionic strength when liposomes contain calcein as well as other polyanions (6‐carboxyfluorescein, sodium citrate).
Infection of neuroblastoma cells by Semliki Forest virus
Authors: VAN STEEG Harry , KASPERAITIS Marcellé , VOORMA Harry O. , BENNE Rob
Published: March 2005

From ribosomal washes of neuroblastoma cells infected with Semliki Forest virus (SFV) a protein of Mr 33000 was purified, which comigrated with the viral capsid protein on sodium dodecyl sulfate/polyacrylamide gels and was recognized by antibodies against the capsid protein of SFV. This protein selectively inhibits the translation of host and early viral 42S mRNA in vitro, but has no effect on late viral 26S and encephalomyocarditis virus mRNA translation. Eukaryotic initiation factor 4B and cap‐binding protein restore the translation of host and 42S mRNA to control levels. The capsid protein specifically prevents the binding of host mRNA into 80S initiation complexes, but has no effect on that of late viral mRNA. We propose that the capsid protein is the component responsible for the shut‐off of host protein synthesis in SFV‐infected cells and for the decreased translational activity of the crude ribosomal washes from these cells.
Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport
Authors: WRIGHT J. Keith , OVERATH Peter
Published: March 2005

The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre‐extraction of the membranes with 5‐sulfosalicylate, solubilization in dodecyl‐O‐β‐d‐maltoside, Ecteola‐column chromatography, and removal of residual impurities by anti‐impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles.

Purification was monitored by tracer N‐[3H]ethylmaleimide‐labeled carrier and by binding of the substrate p‐nitrophenyl‐α‐d‐galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria.

Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain.

Large unilamellar proteoliposomes (1–5‐μm diameter) were prepared from initially small reconstituted vesicles by freeze‐thaw cycles and low‐speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (ΔpH+) or one of its components (ΔΨ or ΔpH).

Comparison of the steady‐state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport.

The half‐saturation constants for active uptake of lactose (KT= 200μM) or β‐d‐galactosyl‐1‐thio‐β‐d‐galactoside (KT= 50–80 μM) by the purified carrier were found to be similar to those measured in cells or cytoplasmic membrane vesicles.

The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat= 3–4s−1 for lactose) but considerably smaller than in cells (kcat= 40–60s−1). Possible reasons for this discrepancy are discussed.
Purification and properties of the basic lectins from winged bean seed [Psophocarpus tetragonolobus(L.) DC]
Authors: KORTT Alexander A.
Published: March 2005

The seeds of winged bean, Psophocarpus tetragonolobus (L.) DC, contain a group of acidic lectins (pI ≊ 5.5) and a group of basic lectins (pI < 9.5) characterised by different erythrocyte hemagglutinating specificities. Three basic lectins were separated and purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP‐Sephadex C‐25. These lectins are glycoproteins with relative molecular mass of 58000. The total carbohydrate content of the major lectins, B2 and B3, was 5% and was comprised of mannose, N‐acetylglucosamine, fucose and xylose in amounts corresponding to 9, 8, 8 and 3 mol/58000g, respectively, for both lectins. Electrophoresis in dodecyl sulfate gave a single subunit of relative molecular mass 29000 for each lectin. Isoelectric focusing in 8 M urea revealed two differently charged subunits in each of the isolated lectins; a total of three different subunits in the three lectins was observed.

The basic lectins have essentially the same amino acid composition and contain no half‐cystine. Two of the lectins (B2 and B3) showed identical amino‐terminal sequences and the sequence of lectin B3 to residue 40 revealed extensive homology with other legume lectins such as soybean lectin.

The basic lectins agglutinated trypsinized rabbit and trypsinized human (type A and B) erythrocytes but not trypsinized human (type O) erythrocytes. The lectins were inhibited by N‐acetylgalactosamine, d‐galactose and d‐galactose containing disaccharides and trisaccharides. The specificity appears to be directed to glycosides having non‐reducing (terminal) α‐d‐galactopyranosyl groups. β‐d‐Galactosides, such as lactose, are poor inhibitors.
Solubilization and reconstitution of α2‐adrenergic receptors from rat and calf brain
Authors: KREMENETZKY Roxana , ATLAS Daphne
Published: March 2005

Sodium cholate and digitonin were used to solubilize α2‐adrenergic receptors from rat and calf brain. Sodium cholate extracted 40–50% of the membrane protein and 25–30% of the binding capacity. Digitonin extracted only 20–30% of the membrane protein and only 10–15% of the binding capacity of the native membranes. Both detergents were removed by dialysis in the presence of phospholipids, and the solubilized protein was precipitated upon addition of poly(ethyleneglycol) and magnesium. In the solubilization/reconstitution process no purification of the α2‐adrenergic receptor was obtained, most probably due to its inactivation by the solubilization conditions. The reconstituted protein(s) tested for binding properties, using p‐[3H]aminoclonidine and/or [3H]clonidine, maintained the pharmacological profile of the native α2‐adrenergic receptor. The potency order of various α2‐agonists and α2‐antagonists as well as their stereoselectivity were identical to those of the native α2‐receptor. Specific receptor binding decreases in the presence of the guanyl nucleotides GTP or guanosine 5′‐[β, γ‐imido]‐triphosphate but not ATP, thus indicating a co‐solubilization of GTP regulatory components (stimulatory protein Ns or inhibitory protein Ni or both). Adenylate cyclase activity of the reconstituted preparation is stimulated threefold by sodium fluoride, suggesting the presence of both Ns‐protein and the catalytic unit (C) in the reconstituted protein(s).
Guinea‐pig casein A cDNA
Authors: HALL Len , LAIRD Jane E. , PASCALL John C. , CRAIG Roger K.
Published: March 2005

The nucleotide sequence (1036 bases) of guinea‐pig casein A mRNA has been determined. Two cDNA recombinant plasmids contained a total of 993 base pairs, including part of the 5′ noncoding region, and the complete coding and 3′ noncoding region. The remaining 5′ noncoding sequence was obtained by primer extension.

The deduced 223‐amino‐acid‐coding sequence of guinea‐pig pre‐casein A exhibited 30% homology with bovine αs2 casein, the most striking similarities being in the locations of potential phosphorylation sites.
Synthesis of components of the cytochrome b‐f complex by isolated pea chloroplasts
Authors: PHILLIPS Andrew L. , GRAY John C.
Published: March 2005

Three components of the cytochrome b‐f complex, cytochrome f, cytochrome b‐563 and a 15.2‐kDa polypeptide, were labelled with radioactive amino acids in isolated pea chloroplasts incubated in the light with added Mg‐ATP. The assembly of cytochrome b‐563 (19.5 kDa) into the cytochrome complex required the presence of added Mg‐ATP whereas cytochrome f (37.3 kDa) and the 15.2‐kDa polypeptide were assembled in its absence. Incorporation of [35S]methionine into the polypeptide chain of cytochrome b‐563 and the 15.2‐kDa component was confirmed by peptide mapping of the products of partial digestion with papain.
The effect of EDTA and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, Methylophilus methylotrophus
Authors: CARVER Mark A. , HUMPHREY Kevin M. , PATCHETT Roy A. , JONES Colin W.
Published: March 2005

The effects of EDTA and related metal‐chelating agents on the respiratory system of the methylotrophic bacterium Methylophilus methylotrophus have been investigated. EDTA completely inhibited whole cell methanol oxidase activity and concomitantly decreased the aerobic steady‐state reduction level of cytochrome c, but only partially inhibited methanol dehydrogenase activity.

Analysis of the inhibition kinetics of EDTA and other chelating agents on methanol oxidase activity indicated that they were effective in the order CDTA < EDTA < HEDTA < EGTA < EDDA, and that they inhibited in an uncompetitive manner. Inhibition by EDTA varied as a function of the ambient cell mass in the assay, and could be partly reversed by the addition of divalent cations. EDTA had no effect on the activity of solubilised methanol dehydrogenase. These and other results indicate that EDTA binds a divalent metal ion, probably Mg2+, which is involved in the functional association of methanol dehydrogenase with the respiratory membrane.

This concept is discussed in terms of the electron transfer properties and spatial organisation of the terminal respiratory chain of M. methylotrophus.
Favism: a hemolytic disease associated with increased superoxide dismutase and decreased glutathione peroxidase activities in red blood cells
Authors: MAVELLI Irene , CIRIOLO Maria Rosa , ROSSI Luisa , MELONI Tullio , FORTELEONI Gavino , DE FLORA Antonio , BENATTI Umberto , MORELLI Alessandro , ROTILIO Giuseppe
Published: March 2005

Red blood cells of favism patients with acute hemolytic crisis have markedly more superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) and less glutathione peroxidase (glutathione: hydrogen‐peroxide oxidoreductase, EC 1.11.1.9) than either normal controls, glucose‐6‐phosphate dehydrogenase‐deficient subjects or favism patients outside hemolytic crisis. This altered value of the two enzyme activities is not due to increased reticulocyte content of blood. The electrophoretic triplet pattern of superoxide dismutase is also changed, with significant increase of the most positively charged band. Similar modifications of the two enzyme activities are observed after treatment of normal red blood cells with high concentrations of divicine and ascorbate, which are redox compounds that are contained in fava seeds. This treatment produces no hemolysis, but leads to hemolysis if the treated cells are resuspended in the homologous plasma. These results suggest a possible role of active oxygen species in the development of favism.
The arrangement of ribosomal RNA genes in Schistosoma mansoni
Authors: SIMPSON Andrew J. G. , DAME John B. , LEWIS Fred A. , McCUTCHAN Thomas F.
Published: March 2005

The two large ribsomal RNA subunits of Schistosoma mansoni are encoded within a 10000‐base sequence, which is tandemly repeated in the schistosome genome. Restriction endonuclease digestion with Bam HI cuts the rRNA gene into three fragments, which have been cloned separately in pBR322 and used to constract a physical map of the gene.

The sequence encoding the smaller rRNA subunit is about 2000 bases in length and is situated on the 5′ sie of the sequence encoding the larger subunit, which is about 4000 bases. Approximately 4000 bases of the rRNA gene are spacer and do not code for mature rRNA. There are approximately 100 copies of the rRNA gene per haploid genome of which about 10% exhibit length heterogeneity, as judged by the hybridization of the rDNA plasmids to restriction endonuclease digests of genomic DNA. These structural variants appear to contain additional DNA suquences at more than one site within the gene and are polymorphic within the species S. mansoni.
Response of Tetrahymena pyriformis to stress induced by starvation
Authors: GALEGO Lisete , BARAHONA Isabel , RODRIGUES‐POUSADA Claudina
Published: March 2005

mRNA synthesis was studied in exponentially growing and starved Tetrahymena pyriformis. Poly(A)‐containing RNAs separated from total RNA by affinity chromatography on oligo(dT)‐cellulose were characterized by polyacrylamide gel electrophoresis; their template activity was assayed in a rabbit reticulocyte lysate system and their translation products were analysed using two‐dimensional electrophoresis according to O'Farrell.

1. Polysome profiles show that the bulk of ribosomes are in 80S monosomes in starved cells, whereas less than 8% are present in the form of monosomes in exponentially growing cells, the rest being engaged in polysomes. Polysomes are almost completely reformed 30 min after addition of enriched medium to suspensions of starved cells. This polysome reformation is dependent on mRNA synthesis since we have shown that it is inhibited by actinomycin D.

2. Electrophoretic profiles of poly(A)‐rich RNA isolated from cytoplasmic fractions of exponential and starved cells are indistinguishable except that in the latter state significant amounts of low‐molecular‐mass species are observed.

3. Poly(A)‐rich RNAs isolated from polysomal and non‐polysomal (subpolysomal) fractions of exponential cells are equally able to promote protein synthesis. The corresponding poly(A)‐rich RNAs isolated from starved cells also possess equal template activities which are, however, 15% lower than those of the poly(A)‐rich RNAs of exponentially growing cells. We also present evidence that in the system used in vitro, polyadenylated RNA isolated from heavy polysomes of starved cells directs the synthesis of four sets of proteins with molecular masses around 100 kDa, 70 kDa, 50 kDa and 30 kDa. The former two groups of proteins are more abundant in the translation products of poly(A)‐rich RNA of starved than of normal cells, whereas the latter two groups are present only in the translation products of poly(A)‐rich RNA of starved cells. The fluorograms of the translation products obtained in vitro from subpolysomal poly(A)‐rich RNA are identical to those obtained from polysomal poly(A)‐rich RNA.

4. Studies on starved cells in vivo show that polypeptides of 100 kDa, 70 kDa and 38 kDa are more strongly labelled and also revealed the specific presence of 85 kDa, 55 kDa, 50 kDa and 25 kDa proteins. These results lead us to the conclusion that this microorganism responds to depleted environmental conditions by regulating gene expression at the transcriptional level, but also at the translational level.
Separation and analysis of novel polyunsaturated mycolic acids from a psychrophilic, acid‐fast bacterium, Gordona aurantiaca
Authors: TOMIYASU Ikuko , YANO Ikuya
Published: March 2005

More than 30 molecular species of highly unsaturated mycolic acids, ranging from C60 to C78 and possessing between two and seven double bonds, have been obtained from a new genus of acid‐fast bacteria, Gordona aurantiaca. They were fully separated and identified as their trimethylsilyl ether derivatives by a combination of silica gel thin‐layer chromatography (TLC), argentation thin‐layer chromatography and gas chromatography/mass spectrometry (GC/MS). On silica gel thin‐layer chromatography two adjacent spots, corresponding to mycolic acids possessing different structures of straight‐chain and α‐alkyl branch, were detected. The lower spot was separated by argentation TLC into four subclasses: monoenoic (including a small amount of saturated), dienoic, trienoic and tetraenoic mycolic acids ranging from C62 to C74, and possessing a C16:0, C18:0, or C20:0 alkyl branch at the C‐2 position. The upper spot was separated by argentation TLC into five subclasses: dienoic (including a small amount of monoenoic), trienoic, tetraenoic, pentaenoic and hexaenoic (heptaenoic) acids ranging from C64 to C78 and possessing a C18:1 or C20:1 alkyl branch at the C‐2 position. These types of mycolic acid structure differ from those reported previously in Mycobacteria and Nocardia, in the numbers of both carbon atoms and double‐bonds and the intermediate length of the α‐alkyl branch. The characteristic polyenoic structure of the straight‐chain alkyl unit was also confirmed by GC/MS analysis of the meromycolaldehydes obtained after pyrolysis of the methyl mycolates. The major aldehydes obtained from the lower‐spot mycolic acids were C44, C46, C48, C50 and C52, centering at C50, while those from the upper‐spot mycolic acids were C48, C50, C52, C54 and C56, centering at C54. These aldehydes were also shown to possess between two and four double bonds in the lower‐spot and between two and seven double bonds in the higher‐spot mycolic acids, respectively. The physiological role of such highly polyunsaturated mycolic acids in psychrophilic acid‐fast bacteria is discussed.
Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross‐linking
Authors: LABURTHE Marc , BREANT Bernadette , ROUYER‐FESSARD Christiane
Published: March 2005

The cleavable cross‐linking reagent dithiobis (succinimidyl propionate) or DTSP was shown to link 125I‐labeled vasoactive intestinal peptide (125I‐VIP) covalently to its receptors in rat intestinal epithelial membranes. DTSP treatment of 125I‐VIP‐labeled membranes inhibited the dissociation of VIP‐receptor complexes in a way which was dependent on both time and concentration (ED50= 200 μM). Polyacrylamide gel electrophoresis of membrane proteins revealed three 125I‐VIP‐protein complexes of Mr 76000, 36000 and 17000. The labeling of those compounds was not observed when: (a) treatment of membranes by DTSP was omitted; (b) the reagent quench, ammonium acetate, was added together with DTSP; (c) DTSP‐treated membranes were incubated with 2‐mercaptoethanol which reduces the disulfide bond present within DTSP. Labeling of Mr‐76000 and Mr‐36000 complexes was specific in that it could be abolished by native VIP, while the labeling of the Mr‐17000 was not. Densitometric scanning of autoradiographs indicated that: (a) labeling of the Mr‐76000 complex was abolished by low VIP concentrations (0.03–10 nM), by VIP agonists with the relative potency VIP > a peptide having N‐terminal histidine and C‐terminal isoleucine amide > secretin, and by GTP (10−5–1 mM) but was unaffected by various other peptide hormones; (b)labeling of the Mr‐36000 complex was inhibited by high VIP concentrations (1–300 nM), by VIP agonists at high concentrations but was not affected by GTP and various peptide hormones. Assuming one molecule of 125I‐VIP was bound per molecule of protein, two proteins with Mr‐73000 and 33000 were identified as VIP binding sites. The Mr‐73000 protein displays many characteristics (affinity, specificity, discriminating power toward agonists, sensitivity to GTP regulation) of the high‐affinity VIP receptors mediating adenylate cyclase activation. The Mr‐33000 protein displays the characteristics (affinity, specificity) of a low‐affinity VIP binding site. This study thus shows the molecular characteristics of the VIP receptor and further argues for the molecular heterogeneity of VIP binding sites.
Structure of the O‐chain of the phenol‐phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9
Authors: CAROFF Martine , BUNDLE David R. , PERRY Malcolm B.
Published: March 2005

The phenol‐phase soluble cellular lipopolysaccharide isolated by the phenol/water extraction method from Yersinia enterocolitica serotpye O:9 cells was shown by hydrolytic, periodate oxidation, methylation and nuclear magnetic resonance studies to be an S‐type lipopolysaccharide with a linear O‐antigenic polysaccharide of 1,2‐linked 4,6‐dideoxy‐4‐formamido‐α‐d‐mannopyranosyl units. The serological cross‐reactivity between Y. enterocolitica serotype O:9 and the lipopolysaccharides of Vibrio cholerae and Brucella species can now be related to the presence of N‐acylated 4‐amino‐4, 6‐dideoxy‐α‐d‐mannopyranosyl residues in their respective O‐antigenic chains.
Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes
Authors: BANFALVI Gaspar , SOOKI‐TOTH Agnes , SARKAR Nilima , CSUZI Sandor , ANTONI Ferenc
Published: March 2005

Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5% dextran T‐150, the four deoxyribonucleoside triphosphates and ATP. Permeable cells could seal the membrane in a serum‐enriched medium within a few hours. 2′‐Deoxycytidine 5′‐triphosphate is effectively substituted by 5‐mercuri‐2′‐deoxycytidine 5′‐triphosphate as a substrate. The newly synthesized mercurated DNA can be separated from cellular DNA and RNA on a thiol‐agarose affinity matrix. The rate of incorporation of [3H]thymidine triphosphate into permeable cells is the same as that of the incorporation of [3H]thymidine into intact cells, corresponding to approximately 30% of the rate in vivo. Synthesis in permeable cells reflects DNA replication shown by inhibitors such as 1‐β‐d‐arabinofuranosylcytosine 5′‐triphosphate (aCTP), nalidixie acid and novobiocin and by density shift experiments. More than 80% of the newly synthesized low‐molecular‐mass DNA, 8–60 nucleotides in length, consists of RNA‐linked DNA. This conclusion is based on phosphorylation with [γ‐32]ATP and polynucleotide kinase and rephosphorylation after alkaline hydrolysis. The 5′ end of RNA consists of adenylate, guanylate, cytidylate and uridylate residues in a ratio of 4:3:1.5:1.5.