bluuebunny/crossref_metadata_2025 huggingface dataset:
The cysteines in position 1 and 86 of rat interferon‐α1 are indispensable for antiviral activity
Authors: Spanjaard Remco A. , van Himbergen John A.J. , van Duin Jan
Published: July 2002

The human, bovine, murine and rat interferon (IFN)‐α families contain 4 conserved cysteines located at positions 1, 29, 99 and 139 that are involved in disulfide bridges. Rat and murine IFN‐α subspecies carry a fifth Cys (Cys‐86) which is not conserved in bovine and human IFN‐α subspecies except for human IFN‐α1. Changing Cys‐86 in rat IFN‐α1, into Ser or Tyr virtually abolished antiviral activity. As shown by others, the substitution of Cys‐86 to Ser in human IFN‐α1 had no pronounced effect on activity. This suggests that in contrast to human and bovine IFN‐α, Cys‐86 in rodent IFN‐α plays a crucial role in receptor binding. Changing Cys‐1 to Gly in rat IFN‐α1 also destroyed activity, in agreement with results obtained in the human IFN‐α1 system.
The phorbol ester‐induced extracellular Ca2+‐independent release of LH is dependent on estradiol and de novo protein synthesis
Authors: Bourne Gregory A. , Das Saswati , Fahmy Nabil W.
Published: July 2002

Phorbol 12‐myristate 13‐acetate (PMA) was used to determine whether the PMA‐induced extracellular Ca2+‐independent release of LH was dependent on sex, estradiol and de novo protein synthesis. Infusions of gonadotropin‐releasing hormone (GnRH) or PMA in a perifusion system stimulated a partial secretion of LH from diestrous II and ovariectomized + estradiol‐treated female pituitaries (responses inhibited by cycloheximide). In contrast, PMA was ineffective in stimulating PRL secretion from these pituitaries, as well as LH secretion from male or ovariectomized female pituitaries. These results indicate that the PMA‐stimulated extracellular Ca2+‐independent secretion of LH is a specific process which is dependent on sex, estradiol and de novo protein synthesis, and mimics the characteristics of the GnRH‐stimulated responses.
Some features of the Streptococcus faecalis Na+‐ATPase resemble thoseof the vacuolar‐type ATPases
Authors: Kakinuma Yoshimi , Igarashi Kazuei
Published: July 2002

In the ethylenediaminetetraacetic acid (EDTA) extract prepared from the membranes of Streptococcus
faecalis, we found the 330‐kDa protein that was coordinately increased with the induction of Na+‐ATPase. It was missed in the EDTA extract of Nakl, a mutant defective in the Na+‐ATPase, but restored in that of its revertant, NaklR. The 330‐kDa protein showed the ATP hydrolytic activity by active staining, and mainly consisted of the polypeptides of 73 kDa, 52 kDa and possibly 38 kDa. In addition, the Na+‐stimulated ATPase of the membranes was sensitive to both nitrate and N‐ethylmaleimide, inhibitors for the vacuolar H+‐ATPase. Thus, the Na+‐ATPase of this organism has a structure similar to vacuolar H+‐ATPase.
Inhibition of photosystem II in Chlorogloeopsis fritschii with shikonin acetate
Authors: Sallal A.-K.J. , Nimer N.A. , Al-Oriquat G.
Published: July 2002

Shikonin acetate extracted from the roots of the desert plant, Arnebia decumbens, was tested for its effect on the photosynthetic electron transport system of C. fritschii. Photosystem (PS) II‐Hill reaction with water and diphenylcarbazide (DPC) as electron donors and ferricyanide as electron acceptor was inhibited completely. Shikonin acetate has little effect on 2,6‐dichlorophenolindophenol (DCPIP)‐Hill reaction and no effect on PSI with durohydroquinone as electron donor. Inhibition of Mehler reaction from water to methylviologen with whole cells was studied using 3‐(3,4‐dichlorophenyl)‐1, 1‐dimethyl urea (DCMU) and shikonin acetate. About 70% inhibition of cyclic and non‐cyclic photophosphorylation was found with 10−7 M shikonin acetate. These results suggest that shikonin acetate inhibits photosynthetic electron flow at the plastoquinone pool.
ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase
Authors: Ackerman Sharon H. , Gatti Domenico L. , Gellefors Pär , Douglas Michael G. , Tzagoloff Alexander
Published: July 2002

The respiratory deficient nuclear mutant of Saccharomyces cerevisiae, N9‐168, assigned to complementation group G95 was previously shown to lack subunit 9, one of the three mitochondrially encoded subunits of the F0 component of the mitochondrial ATPase. As a consequence of the structural defect in F0, the ATPase activity of G95 mutants is not inhibited by rutamycin. The absence of subunit 9 in N9‐168 has been correlated with a lower steady‐state level of its mRNA and an increase in higher molecular weight precursor transcripts. These results suggest that the mutation is most likely to affect either translation of the ??? mRNA or processing or the primary transcript. We have isolated a nuclear gene, designated ATP13, which complements the respiratory defect and restores rutamycin‐sensitive ATPase in G95 mutants. Disruption of ATP13 induces a respiratory deficiency which is not complemented by G95 mutants. The nucleotide sequence of ATP13 indicates a primary translation product with an M??? of 42 897. The protein has a basic amino terminal signal sequence that is cleaved upon import into mitochondria. No significant primary structure homology is detected with any protein in the most recent libraries.
Occurrence of sialidase activity in two distinct and highly homogeneous populations of lysosomes prepared from the brain of developing mouse
Authors: Fiorilli Amelia , Siniscalco Carola , Chiarini Anna , Di Francesco Lucia , Venerando Bruno , Tettamanti Guido
Published: July 2002

Light and heavy lysosomes of mouse forebrain were separated from each other by centrifugation on a Percoll gradient. Light lysosomes were then freed from mitochondria and membranes by sucrose density gradient centrifugation and further purified by floatation‐centrifugation on a sucrose gradient. The final preparations of light and heavy lysosomes, fairly homogenous, carried sinlidase activity, assayed on MU‐NeuAc. The optimal pH was 4.0 and 4.2, the apparent Km
value 2.8 × 10−3 M and 4.2 × 10−3 M and the apparent V
max value 0.11 and 0.47 mU mg−1 protein, for the light and heavy lysosome sialidase, respectively. From 4 days to adulthood the specific activity of the light and heavy lysosome sialidase increased 3‐fold and 1.7‐fold, respectively.
Cultured human keratinocytes cannot metabolize vitamin D3 to 25‐hydroxyvitamin D3
Authors: MacLaughlin Julia A. , Castonguay Maurice A. , Holick Michael F.
Published: July 2002

The metabolism of [3H] vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H] vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high‐performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25‐OH‐D3, 24,25(OH)2D3 were added to the extracts, as markers, prior to HPLC, HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25‐OH‐D3 showed a 10±4% conversion to [3H]1,25,(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25‐OH‐D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25‐OH‐D3.
The conformations of trimethoprim/E. coli dihydrofolate reductase complexes A 15N and 31P NMR study
Authors: Huang Fu-yung , Yang Qing-Xian , Huang Tai-huang , Gelbaum Leslie , Kuyper Lee F.
Published: July 2002

We have employed 15N and 31P NMR techniques to characterize the conformations of trimethoprim (TMP)/E. coli dihydrofolate reductase (DHFR) complexes in the presence and absence of NADPH and NADP+. A single conformation was observed for TMP/DHFR, NADP+/DHFR, NADPH/ DHFR and TMP/NADPH/DHFR complexes. In the ternary complex of TMP/NADP+/DHFR both the 15N and 31P spectra revealed the presence of two conformations. However, the conformations of TMP and NADP+ in the ternary complex may not be correlated, resulting in the possible existence of four conformations for the protein ternary complex.
The primary structure of the Laburnum alpinum seed lectin
Authors: Konami Yukiko , Yamamoto Kazuo , Toyoshima Satoshi , Osawa Toshiaki
Published: July 2002

The complete amino acid sequence of the Laburnum alpinum di‐N‐acetylchitobiose‐binding lectin was determined by using a protein sequencer after digestion with endoproteinases Lys‐C and Asp‐N, and compared with those of other leguminous plant lectins.
Improved band shift assay for the simultaneous analysis of protein—DNA interactions and enzymatic functions of DNA polymerases
Authors: Strick Reiner , Knopf Charles W.
Published: July 2002

A simple method to assay the major properties of DNA polymerases such as template binding, polymerase and exonuclease activities in one step is exemplified with the DNA polymerases of E. coli, bacteriophage T4 and herpes simplex virus. Combining the advantages of the band‐shift assay with the resolving power of polyacrylamide gradient gel electrophoresis, the procedure is particularly useful for a rapid functional analysis of mutant polymerases as well as inhibitors of DNA replication.
The path of a protein chain can be approximated by the conformation dictated by interpeptide ionic bridges
Authors: Lim Valery I. , Venclovas Česlovas , Kurochkina Natalya A.
Published: July 2002

A stereochemical simulation of the formation of ionic bridges between adjacent peptide groups along the polypeptide chain has been made. Such ionic bridges constrain the amino‐acid residues into eight conformations. It is shown that the path of any protein‐chain fragment 10–15 residues long can be approximated well by these conformations. This suggests that the conformations dictated by the ionic bridges can be used as blocks in the formation of the spatial proton structure.
Crystallization of human gelsolin
Authors: McLaughlin P.J. , Gooch J.
Published: July 2002

Human gelsolin has been crystallized by microdialysis techniques to give single crystals that diffract to 3.5 Å resolution. The crystals belong to space group P4212 and have cell dimensions a = 175.0 Å, c = 151.6 Å. They contain two gelsolin molecules in the asymmetric unit.
H7, a protein kinase C inhibitor, increases the glutathione content of neuroblastoma cells
Authors: Romero Francisco J. , Llopis Juan , Felipo Vicente , Miñana Maria-Dolores , Romá Joaquín , Grisolía Santiago
Published: July 2002

It is shown that the intracellular glutathione (GSH) concentration of neuroblastoma‐2a cells in culture increases with a maximum at 24 h after starting treatment with 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), an inhibitor of protein kinase C (PKC). Other inhibitors of this and other protein kinases, e.g. sphingosine, staurosporine, and HA 1004, at the concentrations tested, had a less marked or negligible effect on intracellular GSH concentration. 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA) was also tested and showed no significant effect 24 h after addition.
Protein kinase C activity affects glucose‐induced oscillations in cytoplasmic free Ca2+ in the pancreatic B‐cell
Authors: Kindmark Henrik , Köhler Martin , Efendić Suad , Rorsman Patrik , Larsson Olof , Berggren Per-Olof
Published: July 2002

Acute stimulation of protein kinase C (PKC) inhibited glucose‐induced slow oscillations in cytoplasmic free Ca2+‐concentration, [Ca2+]i, in mouse pancreatic B‐cells. In PKC‐depleted cells glucose induced rapid transients in [Ca2+]i, lasting for approximately 10 s, superimposed on the slow oscillations in [Ca2+]i. It was demonstrated that the transients did not occur in the absence of extracellular Ca2+. Each transient typically was preceded by a slow increase in [Ca2+]i, representing the rising phase of an ordinary glucose‐induced slow oscillation, and the [Ca2+]i, immediately after a transient was lower than just before the spike. These data further emphasize the interplay between voltage‐dependent Ca2+‐channels and the phospholipase C system in the regulation of B‐cell [Ca2+]i‐oscillations.
Neopterin as an endogenous antioxidant
Authors: Kojima Shuji , Icho Takeshi , Kajiwara Yoshio , Kubota Kazuhiko
Published: July 2002

The in vitro potency of neopterin (NP) as an antioxidant and its in vivo activity to suppress alloxan‐induced diabetes were investigated. The reduced form of neopterin, 5,6,7,8‐tetrahydroneopterin (NPH‐4), showed an extremely high superoxide anion radical scavenging activity in two assay systems, i.e. xanthine/xanthine oxidase‐ and macrophage/phorbol myrislate acetate (PMA)‐reaction systems. NPH‐4 also inhibited the oxidation of linoleic acid about as effectively as uric acid. Furthermore, NPH‐4 and NP effectively suppressed alloxan‐induced mouse diabetes. These results suggest that pteridines play an important role as endogenous antioxidant.
The effect of ammonia and pH on brain γ‐glutamyl transpeptidase in young rats
Authors: Št̆astný Frantis̆ek , Pit̆ha Jan , Lisý Václav , Hilgier Wojciech , Kaucká Ivana , Albrecht Jan
Published: July 2002

Acute hyperammonemia, induced by two consecutive injections of ammonium acetate (550 and 450 mg per kg b.wt.), decreased the activity of γ‐glutamyl transpeptidase (GGT) in most brain regions of 18‐ and 30‐day‐old rats. This decrease in the brain GGT activity was more pronounced in younger than in older rats. After the addition or NH4Cl to the incubation medium, the inhibitory action of NH4
+ on this enzyme activity was also demonstrated in crude synaptosomal membranes at pH 7.4, but in a range of NH4
+ concentrations many‐times higher then those found in the plasma or brains of young hyperammonemic rats. Because similar concentrations of NH4
+ stimulated the activity of the purified enzyme from rat kidney (mainly at pH 9.0), the inhibition of GGT activity in the young rat brain is probably mediated indirectly and not by a direct interaction of ammonia with the enzyme molecules.
Air saturation of the medium reduces the rate of phosphorylating oxidation of succinate in isolated mitochondria
Authors: Khazanov V.A. , Poborsky A.N. , Kondrashova M.N.
Published: July 2002

Rat brain mitochondria were isolated and their respiration was polarographically measured without contact with air oxygen. Gas‐saturated experimental mixtures close to the in vivo partial oxygen pressure (normoxic) were compared with the air‐saturated, i.e. hyperoxic, mixtures. The rate of phosphorylating oxidation of added succinate under normoxic conditions was found to be 70–100% higher compared with hyperoxic ones. The addition of succinate dehydrogenase activators results in a more than two‐fold stronger stimulation of succinate oxidation under normoxia than under hyperoxia. Thiol group donors are shown to stimulate respiration under hyperoxia and not under normoxia. Hyperoxic conditions prevented oxidation of the low succinate concentrations corresponding to the physiological ones.
Two subforms of eukaryotic topoisomerase I Purification and structure‐function relationships
Authors: Kudinov Alexei R. , Bronstein Igor B. , Gabibov Alexander G. , Gololobov Gennady V.
Published: July 2002

A new method for isolation of eukaryotic topoisomerase 1 from calf thymus and from Jurkat‐1 cells using HPLC has been developed. The method allows quantitative purification of high molecular weight topo I and of two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions be characterized as two subforms of the enzyme possessing structural and functional differences. The differences in their specific activities, sensitivity to camptothecin and in their proteolytic digestion maps have been demonstrated for the two enzymes.
Role of ADP‐ribosylation in endothelial signal transduction and prostacyclin production
Authors: Halldórsson Haraldur , Bödvarsdóttir Thora , Kjeld Matthías , Thorgeirsson Gudmundur
Published: July 2002

ADP‐ribosylation of proteins by the enzymatic transfer of ADP‐ribose from NAD has been implicated in a number of biological processes. We report that inhibitors of ADP‐ribosylation, most notably the novel inhibitor of arginine specific cellular mono(ADP‐ribosyl) transferase, meta‐iodobenzylguanidine (MIBG) as well as nicotinamide, l‐arginine methyl ester (LAME) and guanyltyramine, inhibit histamine‐induced endothelial production of inositol phosphates, release of arachidonic acid and production of prostacyclin (PGI2). Those same responses were unaffected by MIBG when triggered by thrombin or leukotriene C4. These findings suggest that ADP‐ribosylation serves a role in histamine‐induced production of prostacyclin and imply differences in transduction pathways employed by the different agonists.
Production of recombinant antibodies in lymphoid and non‐lymphoid cells
Authors: Deyev Sergey M. , Lieber Andre , Radko Boris V. , Polanovsky Oleg L.
Published: July 2002

A recombinant tandem of ‘chimeric’ mouse/human immunoglobulin (Ig) genes was constructed and inserted into plasmid pGEM 1 under the control of the T7 bacteriophage RNA polymerase promoter. Lymphoid (Sp2/0) and non‐lymphoid (CHO) cell lines used for transfection contained in their genomes a semisynthetic gene of T7 RNA polymerase and steadily expressed this enzyme. It was shown for the first time that a stable polycystronic transcription of the Ig gene tandem occurs under the control of a single T7 phage promoter, both in lymphoid and non‐lymphoid cells. Synthesis of κ‐light and ε‐heavy Ig chains and functionally active antibodies was observed in the above‐mentioned transfected cell lines.
Stimulated decay of Superoxide caused by ferritin‐bound copper
Authors: Bolann Bjørn J. , Ulvik Rune J.
Published: July 2002

The redox interaction between O2
− and ferritin cannot solely be regarded as a Fe(II) release reaction. We demonstrate that native copper bound to horse spleen ferritin and apoferritin, stimulated the decay of O2
− in a catalytic reaction. Copper was determined by atomic absorption spectrophotometry. Decay of O2
− was monitored spectrophotometrically as the decrease in (A
250‐A
360) at pH 9.5. The catalytic effect was linearly related to the copper content of the protein. Ferritin copper was less efficient than equimolar CuCl2, and iron‐poor ferritin was more efficient than iron‐rich ferritin. The results support a direct antioxidant function of ferritin.
ATP‐dependent protein synthesis in isolated pea chloroplasts
Authors: Taniguchi Michi , Kuroda Hiroshi , Satoh Kimiyuki
Published: July 2002

In the presence of externally added ATP, in the dark, isolated pea chloroplasts accumulate two proteins of molecular masses of about 22 and 24 kDa which precipitate with specific antibodies raised against the D1 protein. By chasing in the light, these proteins disappeared on the fluorogram concomitant with the appearance of the precursor‐ and mature‐sized D1 proteins. Polysome analysis indicated that the 22‐kDa component is associated with membrane‐bound ribosomes and is thus ascribed to a translation intermediate of the D1 protein. On the other hand, the 24‐kDa component could not be found in the polysome fraction under the experimental condition used, suggesting the possibility that this component is a degradation product of the D1 protein. The conclusion from this analysis is that the synthesis and/or stable accumulation of the D1 protein requires factor(s) caused by illumination, in addition to ATP, in isolated pea chloroplasts.
Purification and characterization of p27, a protein from hepatocyte chromatin Evidence suggesting that it binds selectively to guanine‐rich single‐stranded DNA
Authors: Rahat Michal A. , Fry Michael
Published: July 2002

A protein designated p27 that binds preferentially to single‐stranded DNA rich in guanine tracts was purified to near homogeneity from rabbit hepatocyte non‐histone protein extract. Purified p27 migrated as a 27 kDa polypeptide on denaturing SDS‐PAGE and displayed a native molecular mass of ∼ 155 kDa on Sephadex G‐150 or Sepharose 6B‐Cl gel filtration columns. Gel shift analysis indicated that maximum binding of p27 to single‐stranded DNA required the presence of tracts of four or more contiguous guanine residues. The lowest found dissociation constant, 1.4 × 10−8 M/l, was for single‐stranded DNA that contained a (dG)17 run.
Effects of repetitive stimulation, veratridine and ouabain on cytoplasmic pH in frog nerve fibres: role of internal Na+
Authors: Valkina O.N. , Vergun O.V. , Turovetsky V.B. , Khodorov B.I.
Published: July 2002

Changes of cytoplasmic pH (pHi) in frog nerve fibres during repetitive stimulation have been measured using the fluorescent pH indicator dye fluorescein diacetate (FDA). Under control conditions repetitive (10–50 Hz) stimulation caused only a very small decrease in pHi (by 0.015–0.06 pH units). Modification of Na+ channels by veratridine (VER, 10, μM) greatly increased this stimulus‐evoked (SE) internal acidification. Blockade of the Na+‐K+ pump by ouabain (0.5 mM) enhanced the effects VER and prevented pHi recovery after the termination of repetitive stimulation. A similar inhibition of post‐stimulatory recovery of pHi was observed after replacement of external Na+ with Li+, which is not accepted by the Na+‐K+ pump instead of Na+. These data suggest that SE intracellular acidification in nerves results from or is closely associated with an increase in [Na+]i. Treatments that promote Na+ influx and accumulation of Na+ inside the fibre enhance reduction of pHi. Li+ can be substituted for Na+ in this process.
Cotranslational heme binding to nascent globin chains
Authors: Komar Anton A. , Kommer Aigar , Krasheninnikov Igor A. , Spirin Alexander S.
Published: July 2002

Globin synthesis in cell‐free extracts of rabbit reticulocytes was carried out in the presence of 3H‐labeled hemin. Sucrose gradient centrifugation analysis revealed [3H]hemin in the polyribosome fraction. The addition of puromycin resulted in the release of both [3H]hemin‐ and [14C]leucine‐labeled polypeptide from the polyribosomes. The data suggest cotranslational folding of the globin molecule on the ribosome and cotranslational heme binding to the nascent globin chain.
The precursor of the Streptomyces R61 DD‐peptidase containing a C‐terminal extension is inactive
Authors: Fanuel Laurence , Granier Benoît , Wilkin Jean-Marc , Bellefroid-Bourguignon Catherine , Joris Bernard , Knowles Jeremy , Komives Elisabeth , Van Beeumen Jozef , Ghuysen Jean-Marie , Frère Jean-Marie
Published: July 2002

The Streptomyces R61 DD‐peptidase gene encodes a 26‐residue C‐terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active. It also reacted with penicillins significantly more slowly than the mature protein. The introduction of a ‘stop’ codon after that corresponding to the C‐terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E. coli.
The expression of different annexins in the fish embryo is developmentally regulated
Authors: Ivanenkov Vasily V. , Weber Klaus , Gerke Volker
Published: July 2002

The expression of annexins, a family of Ca2+/phospholipid‐binding proteins, was analyzed by biochemical and immunological criteria in the fish Misgurnus fossilis (loach), which is a good model system to study embryonic events. Five different annexins (loach annexins A to E) are present as a maternal pool in the unfertilized eggs. Only annexin E is newly synthesized in the early embryo. Its synthesis, already apparent at mid‐blastula, decreases in later stages when two additional annexins (F and G) appear. They are present among the newly synthesized polypeptides of mid‐gastrula and later embryonic stages and are also found in loach larvae. The developmentally controlled expression of several annexins indicates a specific role of these proteins at certain embryonic stages.
The antioxidant properties of theaflavins and their gallate esters — radical scavengers or metal chelators?
Authors: Miller Nicholas J. , Castelluccio Cinzia , Tijburg Lilian , Rice-Evans Catherine
Published: July 2002

The antioxidant properties of theaflavins and their gallate esters were studied by investigating their abilities to scavenge free radicals in the aqueous and lipophilic phases. The total relative antioxidant activities in the aqueous phase were assessed by measuring their direct ABTS·+ radical scavenging abilities, and by their efficacies in inhibiting the degradation of deoxyribose induced by iron. The propensities for enhancing the resistance of LDL to oxidation mediated by Cu2+ were also measured. The results show that the hierarchy of reactivity of these compounds as antioxidants is: theaflavin digallate > 3′‐monogallate=3‐monogallate > theaflavin. Spectroscopic studies show that all the compounds chelate iron and copper; enhanced absorbance in the visible region is observed in the case of the iron‐digallate complex, but not with copper.
In vitro nuclear reconstitution could be induced in a plant cell‐free system
Authors: Zhao Yun , Liu Xiaoling , Wu Min , Tao Wei , Zhai Zhonghe
Published: July 2002

A cell‐free system derived from carrot cell cytosol extract has been developed for reassembling nuclear structure around the added demembranated sperm chromatin of Xenopus. Morphological evidence suggests that reassembled nuclei display the typical characteristics of normal eukaryotic nuclei, such as double‐layered nuclear membrane and nuclear pores. Micrococcal nuclease treatment indicates that remodeling of the demembranated sperm chromatin has occurred and the structure of nucleosome is formed during nuclear reconstitution. These data indicate that the nuclear reconstitution can be induced in cell‐free systems from plants, and the self‐assembly of the nucleus is ubiquitous in both animal and plant cells.
Crystal structure of the oxidized cytochrome c2 from Blastochloris viridis
Authors: Sogabe Satoshi , Miki Kunio
Published: July 2002

The crystal structure of the oxidized cytochrome c
2 from Blastochloris (formerly Rhodopseudomonas) viridis was determined at 1.9 Å resolution. Structural comparison with the reduced form revealed significant structural changes according to the oxidation state of the heme iron. Slight perturbation of the polypeptide chain backbone was observed, and the secondary structure and the hydrogen patterns between main‐chain atoms were retained. The oxidation state‐dependent conformational shifts were localized in the vicinity of the methionine ligand side and the propionate group of the heme. The conserved segment of the polypeptide chain in cytochrome c and cytochrome c
2 exhibited some degree of mobility, interacting with the heme iron atom by the hydrogen bond network. These results indicate that the movement of the internal water molecule conserved in various c‐type cytochromes drives the adjustments of side‐chain atoms of nearby residue, and the segmental temperature factor changes along the polypeptide chain.
Rab4 affects both recycling and degradative endosomal trafficking
Authors: McCaffrey Mary W. , Bielli Anna , Cantalupo Giuseppina , Mora Silvia , Roberti Vera , Santillo Mariarosaria , Drummond Frances , Bucci Cecilia
Published: July 2002

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen‐1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.
Evidence for a novel regulatory pathway activated by (carba)prostacyclin in preadipose and adipose cells
Authors: Aubert Jérôme , Ailhaud Gérard , Negrel Raymond
Published: July 2002

Prostacyclin, one of the major prostanoids generated in adipose tissue, has been previously described as an autocrine/ paracrine adipogenic effector, acting, in preadipose cells, by means of cAMP and free Ca2+ as cell surface receptor‐mediated messengers. The present study presents evidence for the first time that its stable analogue, carbaprostacyclin, is unique among prostanoids in regulating the expression of two differentiation‐dependent genes in preadipose and adipose cells in a way distinct from that elicited by its cell surface receptor. This regulation is likely mediated by some member(s) of the peroxisome proliferator‐activated receptor family and suggests that prostacyclin behaves as an intracrine effector of adipose cell differentiation.
c‐Fos transrepression revisited
Authors: Cahill Michael A
Published: July 2002

The c‐fos proto‐oncogene was discovered by homology to transforming viral genes, leading to speculation that transforming viruses had captured a cellular gene involved in cell cycle control. Indeed overexpression of c‐Fos protein led to deregulated growth control, and c‐Fos was thought to be so critically involved in cell cycle control that transcriptional transrepression of its own promoter was interpreted as a negative feedback mechanism. However, recent findings render this conclusion improbable, Fos transrepression being most parsimoniously explained as transcriptional squelching imposed by artificially elevated levels of exogenous Fos protein.
Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides
Authors: Dathe Margitta , Wieprecht Torsten , Nikolenko Heike , Handel Liselotte , Maloy W.Lee , MacDonald Dorothy L , Beyermann Michael , Bienert Michael
Published: July 2002

© Federation of European Biochemical Societies.
Deficiency of total collagen content and of deoxypyridinoline in intracranial aneurysm walls
Authors: Gaetani Paolo , Tartara Fulvio , Tancioni Flavio , Rodriguez y Baena Riccardo , Casari Erminia , Alfano Massimo , Grazioli Vittorio
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Purification and structure of mutacin B‐Ny266: a new lantibiotic produced by Streptococcus mutans
Authors: Mota-Meira Marilaine , Lacroix Christophe , LaPointe Gisèle , Lavoie Marc C.
Published: July 2002

Mutacins are bactericidal substances of proteinaceous nature produced by Streptococcus mutans. Lantibiotics are antibacterial substances containing post‐translationally modified amino acids such as lanthionine. Mutacin B‐Ny266 was purified from the cell pellet of S. mutans strain Ny266 by ethanol extraction at pH 2.0 followed by reversed‐phase chromatography (Sep‐Pak® cartridge) and by HPLC on a C18 column. The mean purification factor was 3240±81 and the mean yield was 1.0±0.1%. Molecular mass of mutacin B‐Ny266 as determined by mass spectroscopy is 2270.29±0.21 Da. The amino acid sequence of the purified active fraction was obtained by Edman degradation after treatment with alkaline ethanethiol. Twenty‐one amino acids were detected in this analysis. Mutacin B‐Ny266 belongs to the type A lantibiotics. The proposed sequence is: F–K––W–U–F–––P–G––A–K–O–G––F–N––Y–. The molecule differs from that of epidermin/staphylococcin 1580 and gallidermin at positions 1, 2, 4, 5 and 6.
Natural hidden antibodies reacting with DNA or cardiolipin bind to thymocytes and evoke their death
Authors: Zamulaeva Irina A , Lekakh Irina V , Kiseleva Valentina I , Gabai Vladimir L , Saenko Alexander S , Shevchenko Anatoly S , Poverenny Alexander M
Published: July 2002

Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion‐exchange chromatography and were then affinity‐isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2±10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7±7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine.
The receptor for urokinase‐type plasminogen activator is expressed during mouse spermatogenesis
Authors: Zhou Hongming , Vassalli Jean-Dominique
Published: July 2002

Urokinase‐type plasminogen activator (uPA) binds to a plasma membrane receptor (uPAR) that localizes plasmin generation to the cell environment. Mouse spermatozoa have surface‐bound uPA, which appears to be acquired from genital tract secretions at ejaculation. We determined the presence of uPAR mRNA in spermatogenic cells and their uPA‐binding activity. Northern blot and in situ hybridization demonstrated the presence of uPAR mRNA in germ cells. Binding of uPA, but not of a mutant enzyme lacking the receptor‐binding domain, indicated the presence of uPAR on spermatids and spermatozoa. The uPAR and/or receptor‐bound uPA may be involved in spermatogenesis, spermatozoa maturation or fertilization.
Functional cloning of a cDNA encoding Mei2‐like protein from Arabidopsis thaliana using a fission yeast pheromone receptor deficient mutant
Authors: Hirayama Takashi , Ishida Chika , Kuromori Takashi , Obata Shusei , Shimoda Chikashi , Yamamoto Masayuki , Shinozaki Kazuo , Ohto Chikara
Published: July 2002

To isolate Arabidopsis cDNAs that encode signal transducers and components involved in the regulation of meiosis, a trans‐complementation analysis was performed using a Schizosaccharomyces pombe meiosis‐defective mutant in which the genes for pheromone receptors were disabled. One cDNA obtained in this screening encodes a polypeptide, named AML1, that shows significant similarity to S. pombe Mei2 protein and has three putative RNA‐recognition motifs like as Mei2. Mei2 is involved in the regulation of meiosis in fission yeast. Northern blot analysis showed that the AML1 gene is expressed in each organ. The possible functions of AML1 are discussed.
Mutational analysis eliminates Glu64 and Glu94 as candidates for `catalytic carboxylate' in the bacterial ATP‐binding‐cassette protein MalK
Authors: Stein Anke , Hunke Sabine , Schneider Erwin
Published: July 2002

MalK is the ATP‐hydrolyzing subunit of the binding protein‐dependent ATP‐binding‐cassette (ABC) transport system for maltose from Salmonella typhimurium. In a recent hypothesis, Glu64 and Glu94 of MalK were proposed as candidates for `catalytic carboxylate', common to ATP‐ and GTP‐hydrolyzing proteins [Yoshida and Amano (1995) FEBS Lett. 359, 1–5]. Substitution of both residues and, additionally, Glu74 by either glutamine or glycine and valine, respectively, had no deleterious effect on maltose transport. Thus, our data disprove the above notion.
The selective activation of the cardiac sarcolemmal sodium‐calcium exchanger by plasmalogenic phosphatidic acid produced by phospholipase D
Authors: Hale Calvin C , Ebeling Eric G , Hsu Fong F , Ford David A
Published: July 2002

Since plasmalogens are the predominant phospholipid of cardiac sarcolemma, the activation of the sodium‐calcium exchanger by either plasmenylethanolamine or plasmalogenic phosphatidic acid generated by phospholipase D was explored. Sodium‐calcium exchange activity was 7‐fold greater in proteoliposomes comprised of plasmenylethanolamine compared to proteoliposomes comprised of only plasmenylcholine. Phospholipase D treatment of proteoliposomes resulted in 1 mol % conversion of plasmenylcholine or phosphatidylcholine to their respective phosphatidic acid molecular species with a concomitant 8‐fold or 2‐fold activation of sodium‐calcium exchange activity, respectfully. Thus, phospholipase D‐mediated hydrolysis of plasmalogens to phosphatidic acid may be an important mechanism for the regulation of the sodium‐calcium exchanger.
The role of keratin filaments during nuclear envelope reassembly in Xenopus egg extracts1
Authors: Zhang Bo , Chen Ying , Han Zhiyang , Ris Hans , Zhai Zhonghe
Published: July 2002

We report here a new structure, named `strings‐of‐pearls', which are seen to form in Xenopus egg extracts after incubation, as 200 nm membrane vesicles attach to 10 nm filaments. These membrane vesicles fuse together along the filaments to form annulate lamellae (AL) or attach to demembranated sperm chromatin to initiate assembly of a nuclear envelope. Immunoassay with anti‐keratin antibodies AE3 showed that the filaments were mainly composed of a 56 kDa keratin‐like protein. Addition of AE3 to the extracts resulted in inhibition of AL formation and defective assembly of NE. These results suggest a function of keratins in the assembly of nuclear envelopes during Xenopus development.
Measuring elasticity of biological materials by atomic force microscopy
Authors: Vinckier Anja , Semenza Giorgio
Published: July 2002

Atomic force microscopy (AFM) has proved its value not only for resolving the topographical structure of biological samples, but also for probing inherent properties of biological structures, like local interaction forces, mechanical properties or dynamics in a natural (physiological) environment. This minireview focuses on the acquisition of elasticity data of biomaterials by AFM. A possible theoretical model is presented, followed by a practical `how to do it with AFM', and, finally, a brief overview of publications in this field is given.
Interleukin‐2 stimulates a late increase in phosphatidic acid production in the absence of phospholipase D activation
Authors: Jones David R , Flores Ignacio , Dı́az Ernesto , Martinez-A Carlos , Mérida Isabel
Published: July 2002

The signal transduction pathways involving phospholipid metabolism during T‐cell proliferation remain partly undefined. Herein we show that interleukin‐2 caused a late (>12 h) rise in the intracellular phosphatidic acid content of CTLL‐2 cells which was a consequence of the activation of the enzyme diacylglycerol kinase. No activation of phospholipase D was observed at similar times. Incubation of the cells with a recognized diacylglycerol kinase α isoform inhibitor, R59499, prior to interleukin‐2 stimulation was able to block cell cycle entry, diacyglycerol kinase activation and phosphatidic acid accumulation. In contrast, when R59499 was added 3 h after interleukin‐2, few or no observable effects on the above three parameters were noticed. These results suggest that the early signaling employed by IL‐2 involving the α isoform of diacylglycerol kinase is sufficient to control the late increase in phosphatidic acid and that phosphatidic acid is a mitogenic agent in T‐cells.
The stereochemical course of the reaction mechanism of trehalose phosphorylase from Schizophyllum commune
Authors: Eis Christian , Albert Martin , Dax Karl , Nidetzky Bernd
Published: July 2002

Phosphorolysis of α,α‐trehalose catalyzed by trehalose phosphorylase from the basidiomycete Schizophyllum commune proceeds via net retention of anomeric configuration and yields α‐d‐glucose 1‐phosphate and α‐d‐glucose as the products. In reverse reaction, only the α‐anomers of d‐glucose 1‐phosphate and d‐glucose are utilized as glucosyl donor and acceptor, respectively, and give exclusively the α,α‐product. Trehalose phosphorylase converts α‐d‐glucose 1‐fluoride and phosphate into α‐d‐glucose 1‐phosphate, a reaction requiring the stereospecific protonation of the glucosyl fluoride by a Brønsted acid. The results are discussed with regard to a plausible reaction mechanism of fungal trehalose phosphorylase.
Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2α receptor
Authors: Rehwald Matthias , Neuschäfer-Rube Frank , de Vries Christa , Püschel Gerhard P.
Published: July 2002

For the five principal prostanoids PGD2, PGE2, PGF2α, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G‐protein‐coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His‐81 in the second transmembrane domain of the rat PGF2α receptor, which is conserved among all PGF2α receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site‐directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N‐terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2α with 2‐fold and 25‐fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2α. Wild type receptor and H81Q showed a shallow pH optimum for PGF2α binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2α with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His‐81 in the second transmembrane domain of the PGF2α receptor in concert with Arg‐291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.
A fusion protein of interleukin‐11 and soluble interleukin‐11 receptor acts as a superagonist on cells expressing gp130
Authors: Pflanz Stefan , Tacken Ingrid , Grötzinger Joachim , Jacques Yannick , Dahmen Heike , Heinrich Peter C. , Müller-Newen Gerhard
Published: July 2002

Interleukin‐11 is a hematopoietic cytokine that signals via the signal transducer gp130. Although gp130 is ubiquitously expressed, interleukine‐11 responsiveness is restricted to cells that express the interleukine‐11 receptor α‐subunit. The interleukine‐11 receptor α‐subunit can be functionally replaced by its soluble form indicating that the transmembrane and cytoplasmic parts are not required for signal transduction. Here, we show that a recombinant fusion protein of a fragment of the human interleukine‐11 receptor α‐subunit ectodomain linked to human interleukine‐11 acts as a superagonist on cells expressing gp130 but lacking the membrane‐bound interleukine‐11 receptor α‐subunit. It induces acute phase protein synthesis in hepatoma cells and efficiently promotes proliferation of Ba/F3 cells stably, transfected with gp130. In these bioassays, the fusion protein of a fragment of the human interleukine‐11 receptor α‐subunit ectodomain linked to human interleukine‐11 is 50 times more potent than the combination of interleukine‐11 and the soluble interleukine‐11 receptor α‐subunit. Thus, our findings support the concept that covalent fusion of two soluble proteins required for receptor activation dramatically increases their bioactivity.
The negative charge of Glu‐127 in protein kinase A and its biorecognition
Authors: Batkin Michael , Shaltiel Shmuel
Published: July 2002

A set of mutants of protein kinase A (PKA) in which Glu‐127 was replaced by Gln, Asp, Asn, and Arg was prepared. Their K
m and V
max values show that the negative charge of Glu‐127 (not merely its hydrogen bonding capacity) is indispensable for the kinase activity, since Glu‐127/Gln is inactive, in spite of the fact that it can form hydrogen bonds and is very similar in bulkiness and conformation to wt‐PKA. Glu‐127 is involved in the biorecognition of PKA, interacting ionically with the positively charged guanido group of Arg P‐3 (a major recognition element in the consensus sequence of PKA). In support of this conclusion, it is shown that a regression of the Glu‐127 carboxylate by 1.54 Å (as in Glu‐127/Asp) results in an active kinase with a similar thermal stability and susceptibility to conformation‐dependent proteolysis, a similar V
max, an identical K
m for ATP, but a >20‐fold higher K
m for kemptide. The two inactive mutants of PKA, Glu‐127/Gln and Glu‐127/Asn, are potentially useful for studying protein‐protein interactions of PKA, e.g. for monitoring enzymatically the displacement of active PKA from its complexes.
Demonstration of a novel sulfotransferase in fetal bovine serum, which transfers sulfate to the C6 position of the GalNAc residue in the sequence iduronic acidα1‐3GalNAcβ1‐4iduronic acid in dermatan sulfate
Authors: Nadanaka Satomi , Fujita Masaki , Sugahara Kazuyuki
Published: July 2002

A novel sulfotransferase activity was discovered in fetal bovine serum using pig skin dermatan sulfate as an acceptor and [35S]3′‐phosphoadenosine 5′‐phosphosulfate as a sulfate donor. The enzyme was separated from chondroitin:GalNAc 6‐O‐sulfotransferase by chromatographic techniques. Enzymatic analysis of the reaction products demonstrated that the enzyme transferred sulfate to the C6 position of the GalNAc residue in the sequence ‐iduronic acidα1‐3GalNAcβ1‐4iduronic acid‐. Thus, the enzyme has been identified as a hitherto unreported dermatan sulfate:GalNAc 6‐O‐sulfotransferase. The finding is in sharp contrast to the current concept that in dermatan sulfate biosynthesis GalNAc 4‐O‐sulfation is a prerequisite for iduronic acid formation by C5 epimerase.
The lipoate synthase from Escherichia coli is an iron‐sulfur protein
Authors: Ollagnier-de Choudens Sandrine , Fontecave Marc
Published: July 2002

Lipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV‐visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe‐2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe‐4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed.