bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake and intracellular transport in rat liver of formaldehyde‐treated bovine serum albumin labelled with 125I‐tyramine‐cellobiose
Authors: MISQUITH Sandra , CONINCK Simone WATTIAUX‐DE , WATTIAUX Robert
Published: March 2005

Endocytosis of formaldehyde‐treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I‐tyramine cellobiose (125I‐TCfBSA). 125I‐TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5–10 min after injection and is maintained for up to at least 180 min. During the first 5 min most of radioactivity remains acid‐precipitable. After which, labelled acid‐soluble components are produced at a constant rate for up to 30–40 min.

Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions.

Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma‐membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid‐precipitable and acid‐soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long time (> 30 min) of injection a marked shift of radioactivity distribution towards higher densities occurs.

A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid‐soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid‐precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acidsoluble distribution without having a significant effect on the acid‐precipitable distribution up to 30 min after 125I‐TCfBSA injection.

Glycyl‐1‐phenylalanine‐2‐naphthylamide largely releases acid‐soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I‐TCfBSA injection. On the other hand the proportion of acid‐precipitable radioactivity present in the fraction that can be released is almost zero at 10 min after injection, and it later increases.

The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I‐TCfBSA is quickly delivered to the endosomes. It is then transferred at a constant rate from these organelles to a first population of secondary lysosomes, where it is rapidly hydrolysed. In a later step the hydrolysis products and a small proportion of labelled compounds that remain acid‐precipitable accumulate in a second population of lysosomes.
Purification and characterization of an endonuclease from fruiting caps of basidiomycete Coprinus cinereus
Authors: LU Benjamin C. , WONG William , FANNING Lucia , SAKAGUCHI Kengo
Published: March 2005

An endonuclease was purified from the cap tissues of basidiocarp of Coprinus cinereus collected at early meiotic prophase. It has an optimal activity at pH 7.0 and 37°C. It is a cationic enzyme with a molecular mass of 22 kDa by gel filtration, and contains a 12‐kDa and a 14‐kDa peptide as revealed by SDS gel electrophoresis and Western blot analysis. An antiserum was produced in rabbits against the purified Coprinus endonuclease. The specificity of this antiserum was demonstrated in a dot‐blot analysis and, more critically, in an immunoinhibition of endonuclease activity. The Coprinus endonuclease requires Mg2+ and/or Ca2+ as co‐factors. Ca2+ is more efficient than Mg2+ while the effect of combining both co‐factors is the highest. The Coprinus endonuclease has a substrate preference for single‐strand and supercoiled DNA. It gives only single‐strand nicks on supercoiled DNA at low enzyme concentration and limited time of incubation. At high enzyme concentration and/or long incubation time, double‐strand fragmentation occurred. As is discussed, this endonuclease is believed to be involved in the early phase of meiotic recombination.
Kinetics of inhibition of tissue‐type and urokinase‐type plasminogen activator by plasminogen‐activator inhibitor type 1 and type 2
Authors: THORSEN Sixtus , PHILIPS Malou , SELMER Johan , LECANDER Ingegerd , ÅSTEDT Birger
Published: March 2005

Highly purified plasminogen‐activator inhibitors of type 1 (PAI‐1) and type 2 (PA1‐2), low‐Mr, form, were compared with respect to their kinetics of inhibition of tissue‐type (t‐PA) and urokinase‐type plasminogen activator (u‐PA). The time course of inhibition of plasminogen activator was studied under second‐order or pseudo‐first‐order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t‐PA or u‐PA substrate or by an immunosorbent assay for t‐PA activity. PAI‐1 rapidly reacted with single‐chain t‐PA as well as with two‐chain forms of t‐PA and u‐PA. The second‐order rate constant k for inhibition of single‐chain t‐PA (5.5 × 106M−1s−1) was about three times lower than k for inhibition of the two‐chain activators. PAI‐2 reacted slowly with single‐chain t‐PA, k= 4.6 × 103 M−1 s−1. The association rate was 26 times higher with two‐chain t‐PA and 435 times higher with two‐chain u‐PA. The k values for inhibition of single‐chain t‐PA, two‐chain t‐PA and two‐chain u‐PA were respectively, 1200, 150 and 8.5 times higher with PAI‐1 than with PAI‐2. The removal of the epidermal growth factor domain and the kringle domain from two‐chain u‐PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C‐terminal proteinase part of u‐PA (B chain) is responsible for both the primary and the secondary interactions with PAI‐1 and PAI‐2. The k values for inhibition of single‐chain t‐PA and endogenous t‐PA in plasma by PAI‐1 or PA1‐2 were identical indicating that t‐PA in blood consists mainly in its single‐chain form.
Isolation and structure of a novel pyridine derivative from the Algerian newt, Pleuvodeles waltlii
Authors: YOSHIDA Takeo , IWANAMI Yasuo , BAGNARA Joseph T.
Published: March 2005

Since its discovery more than 20 years ago the structure of a strongly fluorescent compound called “pleurodeles blue” has remained unknown. Isolation of this pigment has been carried out by successive column chromatographies including epichlorohydrin‐triethanolamine‐Sephadex and phospho‐Sephadex. The structural elucidation of a novel pyridone N‐glycoside, 1‐β‐d‐glucopyranosylpyrid‐2(1H)‐one‐6‐acetic acid, is based on a detailed study of its high‐resolution mass spectra, 1H and 13C‐NMR spectra and its hydrolysis to yield d‐glucose.
The role of nucleoside‐diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides
Authors: SEIFERT Roland , ROSENTHAL Walter , SCHULTZ Günter , WIELAND Thomas , GIERSCHICK Peter , JAKOBS Karl. H.
Published: March 2005

NADPH‐oxidase‐catalyzed superoxide (O−2) formation in membranes of HL‐60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL‐60 cytosol. The GTP analogues, guanosine 5′‐[γ‐thio]triphosphate (GTP[γS] and guanosine 5′‐[β,γ‐imido]triphosphate, being potent activators of guanine‐nucleotide‐binding proteins (G proteins), stimulated O−2 formation up to 3.5‐fold. The adenine analogue of GTP[γS], adenosine 5′‐[γ‐thio]triphosphate (ATP[γS]), which can serve as donor of thiophosphoryl groups in kinase‐mediated reactions, stimulated O−2 formation up to 2.5‐fold, whereas the non‐phosphorylating adenosine 5′‐[β,γ‐imido]triphosphate was inactive. The effect of ATP[γS] was half‐maximal at a concentration of 2 μM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[γS]. HL‐60 membranes exhibited nucleoside‐diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[γS] by ATP[γS]. GTP[γS] formation was half‐maximal at a concentration of 3–4 μM ATP[γS] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[γS] on O−2 formation was abolished by the nucleoside‐diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[γS] and considerably diminished stimulation by GTP[γS]. GTP[γS] also served as a thophosphoryl group donor to GDP, with an even higher efficiency than ATP[γS]. Transthiophosphorylation of GDP to GTP[γS] by GTP[γS] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside‐diphosphate kinase.
Studies on the mechanism of translational enhancement by the 5′‐leader sequence of tobacco mosaic virus RNA
Authors: SLEAT David E. , HULL Roger , TURNER Philip C. , WILSON T. Michael A.
Published: March 2005

Translation of foreign mRNAs is enhanced by a cis‐acting derivative (Ω′) of the 5′‐leader sequence (Ω) of tobacco mosaic virus RNA (vulgarestrain). To explain this effect we have conducted several experimentsin vitro.

The presence of various 5′‐terminal sequences, including Ω′, did not significantly increase the half‐lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat‐germ extract. Also, a long leader sequence, unrelated to Ω', did not enhance expression of NPTII mRNAin vitro.

The ability of several leader sequences, including Ω′, to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5′‐leader sequence to enhance translation.

Expression of CAT mRNA in both wheat germ extract and messenger‐dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5′‐proximal Ω′ sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5′‐proximal Ω′ sequence. These data suggest that Ω′ and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5′ terminus more accessible to scanning by 40s ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer‐based secondary‐structure analyses of our SP6 RNA polymerase transcript sequences.

The ability of 5′ leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures.

A different mechanism probably accounts for the Ω′‐dependent enhancement of mRNA expression inEscherichia colior inE. colicell‐free systems.
Analysis of the aromatic 1H‐NMR spectrum of the kringle 5 domain from human plasminogen
Authors: THEWES Theresa , RAMESH Vasudevan , SIMPLACEANU Elena L. , LLINÁS Miguel
Published: March 2005

A kringle 5 domain fragment from human plasminogen has been investigated by 1H‐NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin‐echo experiments and acid/base‐titration studies in conjunction with two‐dimensional double‐quantum and chemical‐shift‐correlated spectroscopies were used to identify individual spin systems. Sequence‐specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in lH2O and in 2H2O have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.
3‐Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism
Authors: CARO L. Heleen P. , PLOMP Peter J. A. M. , WOLVETANG Ernst J. , KERKHOF Christine , MEIJER Alfred J.
Published: March 2005

3‐Methyladenine is generally used as an inhibitor of autophagy [P. 0. Seglen & P. B. Gordon (1982) Proc. Natl Acad. Sci. USA 79, 1889–1892]. Using isolated hepatocytes, we observed that 3‐methyladenine has other effects as well.

3‐Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by CAMP, which slightly increased in the presence of 3‐methyladenine.

Addition of 3‐methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3‐methyladenine in isolated lysosomes.
Insulin and phorbol ester stimulate phosphorylation of acetyl‐CoA carboxylase at similar sites in isolated adipocytes
Authors: HAYSTEAD Timothy A. J. , HARDIE D. Grahame
Published: March 2005

The phorbol ester 12‐0‐tetradecanoyl phorbol 13‐acetate (TPA) stimulates fatty acid synthesis from glucose in isolated adipocytes with a half‐maximal effect at 0.72 μM. In seven batches of cells, the maximal effects of TPA and insulin were 8.5 ± 1.1‐fold and 27.1 ± 2.1‐fold respectively. Insulin also stimulated fatty acid synthesis from acetate 8.9 ± 0.5‐fold (three experiments), but TPA did not significantly increase fatty acid synthesis from this precursor.

In contrast to insulin, TPA treatment of isolated adipocytes did not produce an activation of acetyl‐CoA carboxylase which was detectable in crude cell extracts.

The total phosphate content of acetyl‐CoA carboxylase, isolated from adipocytes in the presence of protein phosphatase inhibitors, was estimated by 32P‐labelling experiments to be 2.6 ± 0.1 (5), 3.4 ± 0.2 (5), and 3.8 ± 0.2 (3) mol/mol subunit for enzyme from control, insulin‐and TPA‐treated cells respectively. Insulin and TPA stimulated phosphorylation within the same two tryptic peptides.

Purified acetyl‐CoA carboxylase is phosphorylated in vitro by protein kinase C at serine residues which are recovered in three tryptic peptides, i.e. peptide T1, which appears to be identical with the peptide Ser‐Ser(P)‐Met‐Ser‐Gly‐Leu‐His‐Leu‐Val‐Lys phosphorylated by cyclic‐AMP‐dependent protein kinase, and peptides Ta and Tb, which have the sequences Ile‐Asp‐Ser(P)‐Gln‐Arg and Lys‐Ile‐Asp‐Ser(P)‐Gln‐Arg respectively, and which appear to be derived from a single site by alternative cleavages. None of these correspond to the peptides whose 32P‐labelling increase in response to insulin or TPA. Peptides Ta/Tb are not significantly phosphorylated in isolated adipocytes, even after insulin or TPA treatment. Peptide T1 is phosphorylated in isolated adipocytes, but this phosphorylation is not altered by insulin or TPA.

These results show that TPA mimics the effect of insulin on phosphorylation, but not activation, of acetyl‐CoA carboxylase, i.e. that these two events can be dissociated. In addition, phorbol ester stimulates phosphorylation of acetyl‐CoA carboxylase in isolated adipocytes, but this is not catalyzed directly by protein kinase C, and acetyl‐CoA carboxylase does not appear to be a physiological substrate for this kinase.
Glycosylation of three molecular forms of human α1‐acid glycoprotein having different interactions with concanavalin A
Authors: BIERHUIZEN Marti F. A. , DE WIT Moniek , GOVERS Carin A. R. L. , FERWERDA Wijnholt , KOELEMAN Carolien , POS Odette , VAN DIJK Willem
Published: March 2005

Human αl‐acid glycoprotein (AGP) was separated into a non‐bound (AGP‐A; 46%), a retarded (AGP‐B; 39%) and a bound fraction (AGP‐C; 150%) using concanavalin A (ConA)‐Sepharose chromatography. The apparent molecular masses, as determined by SDS‐PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively.

The occurrence of N‐linked di‐, tri‐ and tetraantennary glycans on these three molecular forms (AGP‐A, ‐B, and ‐C) was studied by sequential lectin‐affinity chromatography of the 14C‐labelled glycopeptides. These were obtained by extensive pronase treatment followed by N‐[14C]acetylation of the peptide moieties.

The glycopeptides of AGP‐A did not bind to ConA‐Sepharose whereas for AGP‐B and AGP‐C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA‐Sepharose were shown to contain equal amounts of both tri‐ and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L‐PHA).

With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP‐A contains no diantennary structures whereas AGP‐B and AGP‐C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri‐ and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP.

The degree of sialylation of each of the molecular forms was investigated by chromatography on L‐PHA‐agarose and Ricinus communis agglutinin‐I‐agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP‐B and AGP‐C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri‐ and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri‐ and 65% of the tetraantennary chains.

It is suggested that the decrease in the exposure of galactose residues from AGP‐A to AGP‐C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed.
Prokaryotic hopanoids: the biosynthesis of the bacteriohopane skeleton
Authors: FLESCH Gérard , ROHMER Michel
Published: March 2005

Incorporation of 13C‐labelled acetate into the hopanoids of the purple non‐sulfur bacteria Rhodopseudomonas palustris and Rhodopseudomonas acidophila and the facultative methylotroph Methylobacterium organophilum showed that the bacteriohopane skeleton is built from an unique carbon/carbon linkage between the triterpenic hopane moiety and the C‐5 carbon of a d‐ribose derivative arising from the non‐oxidative pentose phosphate pathway.

Furthermore a probable compartmentation of the acetate metabolism could be observed in these bacteria. Whereas exogenous acetate was directly incorporated into the glucose derivatives and poly‐(3‐hydroxybutyrate), the isoprenic units were apparently solely synthesized from two acetate units arising from the glyoxylate cycle and a third one issued either from the glyoxylate cycle or from the Entner‐Doudoroff pathway of glucose catabolism. Although an unknown biosynthetic pathway different from that usually proposed for isoprenoid biosynthesis can not be excluded, the former hypothesis explained all labelling patterns observed on the triterpenic skeleton.
Bordetella pertussis adenylate cyclase
Authors: GENTILE Fabrizio , RAPTIS Anastassios , KNIPLING Leslie G. , WOLFF J.
Published: March 2005

Exposure of Chinese hamster ovary, mouse adrenal cortex tumor (Y‐1), THP‐1 and U‐937 cells and human erythrocytes to adenylate‐cyclase‐containing urea extracts of Bordetella pertussis (strain 114) organisms promotes the formation of large concentrations of intracellular CAMP. Accumulation is dependent on dose and temperature, with significant accumulation occurring at 4° C, and is virtually instantaneous, with a doubling at 1 min. There is an absolute Ca2+ requirement but external calmodulin (the activator of cyclase activity) has no effect except in erythrocytes and U‐937 cells, where it reduces CAMP accumulation. However, calmodulin antagonists inhibit cAMP accumulation. In Y‐1 adrenal cells the urea‐extract adenylate cyclase stimulates steroidogenesis.

Anti‐(B. pertussis) antibodies inhibit cyclase activity and prevent further cAMP accumulation after 10 min in cells previously exposed to urea extract. The same effect is obtained by washing. This suggests that a portion of the cyclase is associated with cells in a form not accessible to antibody or washing but accessible to substrate, which we interpret as internalized enzyme with a short lifetime. Continuing CAMP accumulation thus appears to need a continuing source of external cyclase.

Inhibitors of the effect of diphtheria toxin, such as NH4Cl, methylamine, chloroquin or monensin, have no inhibitory effect on the accumulation of intracelluar cAMP promoted by the internalized adenylate cyclase of urea extracts of B. pertussis organisms. We conclude that entry of the cyclase into cells is not by receptor‐mediated endocytosis.
Single‐cell analysis of the intracellular pH and its regulation during the monocytic differentiation of U937 human leukemic cells
Authors: LADOUX Annie , MIGLIERINA Roger , KRAWICE Irène , CRAGOE Edward J. , ABITA Jean Pierre , FRELIN Christian
Published: March 2005

Monocytic differentiation of U937 cells induced by retinoic acid is accompanied by a 0.2‐pH‐unit cell alkalinisation. The effect of retinoic acid on intracellular pH (pHi) develops slowly and it precedes the differentiation of the cells by 24 h. Heterogeneity in cellular pHi values was assessed using flow cytometry. It was higher at the differentiated stage than at the undifferentiated stage. It was reduced under conditions of clamped pHi values. Two membrane mechanisms allow U937 cells to recover from an intracellular acidosis. These are the Na+/H+ exchange system and a Na+‐dependent HCO3−/Cl− exchange system. The increase in the pHi observed after monocytic differentiation resulted from a twofold increase in the maximum activity of the Na+/H+ exchange system with no change in the activity of the bicarbonate‐dependent system. The properties of interaction of the Na+/H+ exchanger of U937 cells with Na+, Li+, amiloride and its derivatives were defined and appeared to be unique to human leukemic cells.
Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade
Authors: TAKADA Haruhiko , KOTANI Shozo , TANAKA Shigenori , OGAWA Tomohiko , TAKAHASHI Ichiro , TSUJIMOTO Masachika , KOMURO Tetsuo , SHIBA Tetsuo , KUSUMOTO Shoichi , KUSUNOSE Naoto , HASEGAWA Akira , KISO Makoto
Published: March 2005

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate.(β1‐6)‐d‐Glocosamine disaccharide bisphosphates, which had neither 3‐hydroxyacyl nor 3‐acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non‐reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3‐hydroxyacyl groups on the bisphosphorylated (β1‐6)‐d‐glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3‐acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity.

Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non‐hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non‐reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.
Displacement of histones by sperm‐specific proteins at different stages of spermatogenesis of squid
Authors: KADURA Sergey N. , KHRAPUNOV Sergey N.
Published: March 2005

Changes in the composition of the chromatin basic proteins during spermatogenesis of the squid Illex argentinus were studied. The core histones of I. argentinus slightly differ from those of calf thymus in the subfractional composition of histones H2A and H2B.

A similar amino acid composition is revealed in the histones H1 of the squid I. argentinus and calf thymus. Histone H1 of the squid has a lower molecular mass and a special subfractional composition as compared to those of calf thymus, grass carp and carp studied formerly [Kadura et al. (1983) Comp. Biochem. Physiol. 743, 343–3501. Neither the fractional nor subfracitonal composition of histones changes during spermatogenesis. The two new proteins were revealed in the chromatin composition of squid testes and spermatozoa illexines I1 and I2. Illexine I2 is composed of two subfractions I2‐1 and I2‐2. Illexine I2 shows a high content of arginine (75 mol/100 mol). Serine (10 mol/100 mol), histidine (3,2 mol/100 mol) and tyrosine residues (2,9 mol/100 mol) are also present. Illexine I1 shows the presence of arginine (45,6 mol/100 mol), lysine (7.6 mol/100 mol), serine (11.4 mol/100 mol), hystidine (2.3 mol/100 mol) and tyrosine residues (2.8 mol/100 mol).

Molecular masses of illexines I2 and I1 are approximately 7 kDa and 9 kDa respectively. It is supposed that during spermatogenesis the histones are displaced in two‐stage order: histones → I1→ I2.
Rearrangements of chromatin structure during spermatogenesis of squid
Authors: KHRAPUNOV Sergey N. , KADURA Sergey N. , DRAGAN Anatol I.
Published: March 2005

A step wise replacement of somatic histones on sperm‐specific proteins (we have termed them illexines I1 and I2) is found to occur during spermatogenesis of squid Illex argentinus [Kadura, S. N. and Khrapunov, S. N. (1988) Eur. J. Biochem. 175, 603–607]. The chromatin from nuclei of squid immature testes has a nucleosomal DN A repeat which corresponds to the nucleosomal repeat of calf thymus chromatin (195 ± 5 bp). As spermatozoa become mature and illexine I2 accumulates in the chromatin, the nucleosomal structure of the latter disappears and chromatin compacting takes place. The chromatin DNA from squid spermatozoa is highly resistant to micrococcal nuclease action.

Spectrophotometry and spectrofluorimetry were to establish that neither illexine I1 nor illexine I2 forms a globular structure in solution under any conditions studied. Illexine I2 (approx. 7 kDa) shows a high affinity to DNA and remains bound to it under conditions when complexes of illexine I1 (approx. 9 kDa) and salmine (approx. 4.5 kDa) with DNA completely dissociate. This fact, allowing for a similar content (about 75%) of arginine in illexine I2 and salmine, suggests high clustering of arginine residues in the composition of illexine I2.

It is suggested that the initial stage of histone substitution with illexine I1, which has a more moderate affinity to DNA than illexine I2, prepares chromatin for the formation of a highly packed structure by illexine I2 during squid spermatogenesis.
Biliverdin IXδ and neobiliverdin IXδ, isolated from the ovaries of the marine snail, Turbo cornutus
Authors: BENEDIKT Eva , GOSSAUER Albert , KÖST Hans‐Peter , MIKI Wataru , YAMAGUCHI Katsumi
Published: March 2005

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (=32‐hydroxy‐mesobiliverdin IXα), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IXδ and neobiliverdin IXδ. This is the first report of naturally occurring bile pigments with IXδ structure.
A mathematical model of the Calvin photosynthesis cycle
Authors: PETTERSSON Gosta , RYDE‐PETTERSSON Ulf
Published: March 2005

A mathematical model is presented for photosynthetic carbohydrate formation in C3 plants under conditions of light and carbon dioxide saturation. The model considers reactions of the Calvin cycle with triose phosphate export and starch production as main output processes, and treats concentrations of NADPH, NAD+, CO2, and H+ as fixed parameters of the system. Using equilibrium approximations for all reaction steps close to equilibrium, steady‐state and transient‐state relationships are derived which may be used for calculation of reaction fluxes and concentrations of the 13 carbohydrate cycle intermediates, glucose 6‐phosphate, glucose 1‐phosphate, ATP, ADP, and inorganic (Ortho)phosphate.

Predictions of the model were examined with the assumption that photosynthate export from the chloroplast occurs to a medium containing orthophosphate as the only exchangeable metabolite. The results indicate that the Calvin cycle may operate in a single dynamically stable steady state when the external concentration of orthophosphate does not exceed 1.9 mM. At higher concentrations of the external metabolite, the reaction system exhibits overload breakdown; the excessive rate of photosynthate export deprives the system of cycle intermediates such that the cycle activity progressively approaches zero.

Reactant concentrations calculated for the stable steady state that may obtain are in satisfactory agreement with those observed experimentally, and the model accounts with surprising accuracy for experimentally observed effects of external orthophosphate on the steady‐state cycle activity and rate of starch production.

Control analyses are reported which show that most of the non‐equilibrium enzymes in the system have a strong regulatory influence on the steady‐state level of all of the cycle intermediates. Substrate concentration control coefficients for cycle enzymes may be positive, such that an increase in activity of an enzyme may raise the steady‐state concentration of the substrate is consumes.

Under optimal external conditions (0.15–0.5 mM orthophosphate), reaction flux in the Calvin cycle is controlled mainly by ATP synthetase and sedoheptulose bisphosphatase; the cycle activity approaches the maximum velocity that can be supported by the latter enzyme. At lower concentrations of external orthophosphate the cycle activity is controlled almost exclusively by the phosphate translocator. At high external orthophosphate concentrations the phosphate translocator resumes predominant control, but also other non‐equilibrium enzymes gain strong flux control with one notable exception: ribulosebisphosphate carboxylase has no significant regulatory influence on the cycle activity under conditions of light and CO2 saturation, nor does it control the concentration of any cycle intermediate other than its substrate.
Microsomal and cytosolic epoxide hydrolases, the peroxisomal fatty acid β‐oxidation system and catalase
Authors: STEINBERG Pablo , SCHLADT Ludwig , DIENES Hans Peter , TIMMS Christopher , OESCH Franz
Published: March 2005

A number of structurally unrelated hypolipidaemic agents and certain phthalate‐ester plasticizers induce hepatomegaly and proliferation of peroxisomes in rodent liver, but there is relatively limited data regarding the specific effects of these drugs on liver non‐parenchymal cells. In the present study, liver parenchymal, Kupffer and endothelial cells from untreated and fenofibrate‐fed rats were isolated and the activities of two enzymes associated with peroxisomes (catalase and the peroxisomal fatty acid P‐oxidation system) as well as cytosolic and microsomal epoxide hydrolase were measured.

Microsomal epoxide hydrolase, cytosoic epoxide hydrolase and catalase activities were 7 – 12‐fold higher in parenchymal cells than in Kupffer or endothelial cells from untreated rats; the peroxisomal fatty acid β‐oxidation activity was only detected in parenchymal cells. Fenofibrate increased catalase, cytosolic epoxide hydrolase and peroxisomal fatty acid β‐oxidation activities in parenchymal cells by about 1.5, 3.5‐ and 20‐fold, respectively. The induction of catalase (2 ‐ 3‐fold) and cytosolic epoxide hydrolase (3 ‐ 5‐fold) was also observed in Kupffer and endothelial cells; furthermore, a low peroxisomal fatty acid β‐oxidation activity was detected in endothelial cells. Morphological examination by electron microscopy showed that peroxisomes were confined to liver parenchymal cells in untreated animals, but could also be observed in endothelial cells after administration of fenofibrate.
Chorismate mutase:prephcnate dehydratase from Acinetobacter calcoaceticus
Authors: AHMAD Suhail , WILSON Anna‐Tan , JENSEN Roy A.
Published: March 2005

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91000) composed of 45‐kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L‐phenylalanine (inhibitor of both activities) or L‐tyrosine (activator of the dehydratase activity) during gel filtration.

Chorismate mutase activity followed Michaelis‐Menten kinetics with a Km of 0.55 mM for chorismate. L‐Phenylalanine was a relatively poor non‐competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate‐saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate‐binding sites. L‐Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L‐Phenylalanine inhibited the dehydratase activity, and the substrate‐saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L‐phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L‐phenylalanine gave Hill plots having a slope of ‐ 2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate‐saturation curves were changed to near‐hyperbolic form. The happ values decreased with increase in ionic strength.

Antibodies raised against the purified P protein showed cross‐reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross‐reaction was superior with P protein from Neisseria gonorrhaeae than with that from the more closely related Escherichia coli.
Monomerization of cytochrome oxidase may be essential for the removal of subunit III
Authors: FINEL Moshe , WIKSTRÖM Mårten
Published: March 2005

Incubation of cytochrome oxidase, under conditionsused as intial steps in treatment to remove sub‐unit III, causes at least partial monomerization of the enzyme.

The extent of removal of subunit III by anion‐exchange fast protein liquid chromatography (FPLC) is much increased if the enzyme is fully monomerized before it is applied to the column.

Subunit III is incompletely removed by chymotrypsin treatment. A digestion product of subunit III migrating in SDS‐PAGE like subunit IV, is detected with specific antibodies. The amount of this product is reduced when monomerization is increased by raising the detergent/protein ration.

The results suggest that monomerization facilities removal of subunit III and exposes it to further chymotrypsin digestion. We propose that subunit III is at least in part located in the junction between the monoments in the cytochrome oxidase dimer.
Forskolin alters sensitivity of the cAMP‐generating system to stimulatory as well as to inhibitory agonists
Authors: MOKHTARI Abdel Rhani , DO KHAC Lien , HARBON Simone
Published: March 2005

In both the intact guinea pig myometrium and human platelets, cAMP accumulation was enhanced by prostagladin I2 (prostacyclin, PGI2) and forskolin caused rises in platelet cAMP concentration though a single low‐affinity ineraction (Kapp= 90 μ) while in myometrium, activation involved both a low‐affinity (Kapp= 10 μ) and a high‐affinity (Kapp= 0.8 μM) component. The contribution of the high‐affinity component could be reduced when endogenous PGI2 was decreased. In both tissuse, the synergistic effect of forkolin in the presence of PGI2 was mediated by a single high‐affinity ineraction (Kapp= 0.3μM). The data were consistent with a low‐affinity interaction of the diterpene with the cyclase catalytic unit C generation C… forskolin state and with a hogh‐affinity interaction of the diterpene with the activated complex (stimulatory regulatory protein) and C generation the potentiated Gs‐C… forskolin state.

Bothe norepinephrine in platelets and carbachol in the myometrium)via Gi, the inhibitory regulatory protein)inhibited PGI2‐mediated cAMP accumulation (EC50= 100 nM and 8 nM respectively). The persistently activated cAMP‐generation system induced by choleratoxin in the myometrium was also suceptible to inhibition but the EC50 for carbachol was increased to 50 nM and the extent of inhibition was decreased. Forskolin‐mediated effect in platelets was inhibited by norepinephrine aswas the PGI2 response. By contrast, the synergistic state of the cyclase resisted the inhibitory action of norepirne and carbachol in platelets and myometrium repectively. In the myometrium, where the cAMP response due to forskolin alone partially involved some synergistic Gs‐C… forskolin species, carbachol at 50 μM elicited no more than 30% inhibition. Inhibition was partly improved (60% inhibition at 1 μM carbachol)when the contribution of the Gs‐C species was decreased by lowering the concertration of lacal PGI2. Partial inhibition by norepinephrine was similarly observed in platelets under partial synergistic condition. The data suggest that recepto‐mediated inhibition of cAMP generation could be differentially expressed depending on the nature of the active species of the cyclase involved in the stimulatory responses.
A human glioma cell line secretes three structurally and functionally different dimeric forms of platelet‐derived growth factor
Authors: HAMMACHER Annet , NISTÉR Monica , WESTERMARK Bengt , HELDIN Carl‐Henrik
Published: March 2005

A human malignant glioma cell line, U‐343 MGa Cl 2:6, has previously been shown to secrete platelet‐derived‐growth‐factor(PDGF)‐like activity [Nistér, M., Heldin, C.‐H., Wasteson, Å. and Westermark, B. (1984) Proc. Nail Acad. Sci. USA 81, 926‐930]. We report here that this activity consists of three different molecules separable by reversed‐phase chromatography and immobilized‐metal‐ion affinity chromatography. HPLC reversed‐phase chromatography resolved two peaks of activity, which were denoted glioma‐derived growth factor‐I (GDGF‐I) and GDGF‐II. GDGF‐I was purified to >90% purity; in SDS gel electrophoresis, it appeared as a 31‐kDa component which by reduction was converted to 17 kDa. Immunoprecipitation of radiolabeled, reduced and alkylated GDGF‐I with antisera made against peptides from the A and B chains of PDGF, gave a specific signal only with antiserum against the A chain. Furthermore, when reduced and alkylated GDGF‐I was analyzed by reversed‐phase HPLC, it eluted at the position of PDGF A chains. We conclude that GDGF‐I is a homodimer of a polypeptide similar to the A chain of PGDF. GDGF‐II was found to have higher mitogenic activity than GDGF‐I. Analysis by immunoprecipitation with PDGF‐chain‐specific antisera revealed that GDGF‐II contained a polypeptide similar to the B chain of PDGF. Immobilized‐metal‐ion affinity chromatography revealed that 95% of the mitogenic activity of GDGF‐II consisted of a heterodimer of one A and one B chain, whereas 5% consisted of a B‐chain homodimer. Thus, U‐343 MGa Cl 2:6 cells secrete all three possible dimeric forms of PDGF.
Murine hybridoma/plasmacytoma growth factor
Authors: SIMPSON Richar J. , MORITZ Robert L. , RUBIRA Michael R. , VAN SNICK Jacques
Published: March 2005

Murine interleukin‐HP1 (HP1) was originally identified as a T‐cell derived lymphokine with growth factor activity for B‐cell hybridomas and plasmacytomas. This growth factor was recently shown to stimulate both normal B‐cell differentiation and T‐cell growth factor activity. We have determined the complete amino acid sequence of HP1 on 40 μg (approximately 2 nmol) protein using a combination of sensitive microbore column (1.0 and 2.1 mm internaldiameter) HPLC, peptide mapping and automated amino acid microsequence analysis. Ion‐pairing chromatography was employed to isolate hydrophilic peptides which were not retained on conventional reversed‐phase HPLC systems. The molecule consists of 187 amino acid residues with a calculated molecular mass of 21710 Da.

Although there is virtually no similarity between the NH2‐terminal region of HP1 and its human biological counterpart (26‐kDa protein/interferon‐β2= B‐cell stimulatory factor‐2/interleukin‐6), these studies demonstrate extensive amino acid similarity inthe middle and COOH‐teminal regions of these molecules suggesting that HP1 is the murine homologue of human interleukin‐6.
A new family of repetitive, retroposon‐like sequences in the genome of the rainbow trout
Authors: WINKFEIN Robert J. , MOIR Robert D. , KARWETZ Stephen A. , BLANCO Jeronimo , STATES J. Christopher , DIXON Gordon H.
Published: March 2005

We have identified a new family of interspersed, moderately repetitive DNa elements, termed the RSf‐1 family, in the genome of the rainbow trout. Two of the elements examined here are situated upstream of sequences which code for trout nuclear proteins; a protamine gene (p101) and the clustered histone H4 gene. Sequence comparision of various RSg‐1 elements indicated a high degree of nucletide sequence homology between different members of the family. These repetitive elements exhibit well defined 3′ ends which contain poly (A) tract also conform to a consensus sequence. A similar sequence is also found flanking the 5′ terminus of the element in the protamine clone p101, and thus may represents a target‐site duplication generatedupon insertion of the element into the genome. These carachteristics, together with the heterogeneous nature of the 5′ ends of the elements, are reminiscent of processed pseudogenes and retroposons such as the mammalian L1 family of interspersed repetitive elements.
Diethylstilbestrol
Authors: STRID Åke , NYRÉN Pål , BALTSCHEFFSKY Margareta
Published: March 2005

The hydrophobic compound diethylstilbestrol inhibits the generation of the proton gradient and the membrane potential in chromatophores from Rhodospirillum rubum and dissipates proton gradients over asolectin vesicle membranes. The Ca2+‐ATPase activity of chromatophores, of purified F0F1‐ATPase and of purified F1‐ATPase is also decreased in the presence of diethylstilbestrol. Other repressed activities are the pyrophosphatase activity of soluble pyrophosphatase from yeast and the NADH oxidation by L‐lactate: NAD oxidoreductase. We have previously reported that also ATP synthesis, PPi synthesis and PPi hydrolysis of R. rubrum chromatophores are inhibited by diethylsilbestrol [Strid et al. (1987) Biochim. Biophys. Avta 892, 236‐244], Addition of bovine serum albumin reverses or prevents diethylstilbestrol‐induced inhibition of the activities tested. On the other hand, the Mg2+‐ATPase activity of chromatophores, purified F0F1‐ATPase and purified F1ATPase are stimulated by low concentrations of diethylstilbestrol. On the basis of its hydrophobicity and the reversal of its inhibition by bovine serum albumin, diethylstilbestrol is proposed to act unspecifically on membranes and at hydrophobic domains of proteins. Such an attack upon the subunits of the F1‐ATPase, altering the subunit interactions, is proposed to explain the different results obtained for the Ca2+‐ATPase and the Mg2+‐ATPase.
Differential inhibition of various mammalian DNA polymerase activities by ammonium 21‐tungsto‐9‐antimoniate (HPA23)
Authors: ONO Katsuhiko , NAKANE Hideo , BARRÉ‐SINOUSSI Françoise , CHERMANN Jean‐Claude
Published: March 2005

The 21‐tungsto‐9‐antimoniate ammonium salt (HPA23), known as an antiviral agent, has been shown to be a potent inhibitor of both human and murine DNA polymerase α and murine DNA polymerase γ. HPA23 inhibited the activity of DNA polymerase α in noncompetitive fashion with respect to either deoxynucleotide substrate or nucleic acid template · primer. The Ki of murine DNA polymerase α for HPA23 was determined to be 24 nM. The activity of mouse DNA polymerase γ also was strongly inhibited by HPA23 (Ki, 20 nM), and the mode of inhibition was competitive with respect to the template · primer, (rA)n· (dT)12‐18, and noncompetitive to substrate, dTTP. DNA polymerase β and terminal deoxynucleotidyltransferase, however, were relatively resistant to inhibition by HPA23. The observed inhibitions by HPA23 seem to be closely related to the polyanionic property of this drug.
Similarity of the E1 subunits of branched‐chain‐oxoacid dehydrogenase from Pseudomonas putida to the corresponding subunits of mammalian branched‐chain‐oxoacid and pyruvate dehydrogenases
Authors: BURNS Gayle , BROWN Tracy , HATTER Kenneth , IDRISS John M. , SOKATCH John R.
Published: March 2005

The genes encoding proteins responsible for activity of the E1 component of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida have been subcloned and the nuckotide sequence of this region determined. Open reading frames encoding E1α (bkdA1, 1233 bp) and E1β (bkdA2, 1020 bp) were identified with the aid of the N‐terminal sequence of the purified subunits. The Mr of E1α was 45158 and of E1β was 37007, both calculated without N‐terminal methionine. The deduced amino acid sequences of E1α and E1β had no similarity to the published sequences of the E1 subunits of pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli. However, there was substantial similarity between the E1α subunits of Pseudomonas and rat liver branched‐chain‐oxoacid dehydrogenases. In particular, the region of the E1α subunit of the mammalian branched‐chain‐oxoacid dehydrogenase which is phosphorylated, was found to be highly conserved in the Pseudomonas E1α subunit. There was also considerable similarity between the E1β subunits of Pseudomonas branched‐chain‐oxoacid dehydrogenase and human pyruvate dehydrogenase.