bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimation of the functional size of Photosystem II
Authors: Nugent J.H.A. , Atkinson Y.E.
Published: July 2002

The radiation inactivation technique was used to measure the functional size of Photosystem II from Phormidium laminosum. Using frozen samples irradiated in liquid nitrogen, the technique was shown to give reproducible results. The mean functional size of PS II was found to be 125 kDa in a range from 99 kDa to 160 kDa. Comparing this result to those reported in other studies using SDS—PAGE suggests this functional size could be composed of one 52‐kDa reaction centre polypeptide, one 33‐kDa herbicide binding protein, and one 34‐kDa polypeptide associated with oxygen evolution. Even the maximum limit of the estimate would only allow the further inclusion of approximately 40 kDa of polypeptide. The result suggests a smaller and simpler composition for PS II than indicated by SDS—PAGE.
Purification and characterization of a plasmid‐encoded aminoglycoside‐(3)‐N‐acetyltransferase IV from Escherichia coli
Authors: Bräu Barbara , Piepersberg Wolfgang
Published: July 2002

Plasmid‐encoded aminoglycoside‐(3)‐N‐acetyltransferase IV, AAC(3)‐IV, was purified to homogeneity by affinity chromatography from E. coli The enzyme was shown to consist of a monomer, with the apparent M
r being in agreement with that calculated from the nucleotide sequence of the aacC4 gene (M
r 28 500). Determination of the sequence of the N‐terminal 6 amino acids revealed that processing did not occur, indicating the cytoplasmic localization of the AAC(3)‐IV enzyme. A correlation of antibiotic resistance with K
m values of the purified enzyme for a corresponding set of aminoglycoside substrates is discussed with respect to the mechanism of resistance in vivo.
Cloning and sequencing of the Pseudomonas aeruginosa PAK pilin gene
Authors: Pasloske Brittan L. , Finlay B.Brett , Paranchych William
Published: July 2002

A 1.2‐kilobase (kb) HindIII restriction fragment containing the pilin gene from Pseudomonas aeruginosa PAK has been cloned and sequenced. The pilin protein is 144 amino acids in length with a positively charged leader sequence of 6 amino acids. There is probably only one copy of the gene per chromosome.
Bile acid binding in plasma: the importance of lipoproteins
Authors: Salvioli G. , Lugli R. , Pradelli J.M. , Gigliotti G.
Published: July 2002

Bile acid (BA) hydrophobicity, evaluated by the octanol‐water partition coefficient, decreases along the series deoxycholic acid ‐ chenodeoxycholic acid ‐ hyodeoxycholic acid ‐ ursodeoxycholic acid ‐ cholic acid (CA) ‐ ursocholic acid (UCA). In vitro experiments carried out using dialysis techniques (to determine the maximum BA binding) and ultrafiltration of plasma pre‐incubated with 0.1 mM BA (to assess the distribution of BA between the different lipoprotein fractions) showed that the maximum binding of BA to plasma and lipoproteins follows the same order of hydrophobicity. The fraction not bound to proteins, greater with the hydrophilic BA (UCA and CA), is distributed in the lipoprotein fractions and in particular in high density lipoproteins.
Hydroxylation of acetone by ethanol‐ and acetone‐inducible cytochrome P‐450 in liver microsomes and reconstituted membranes
Authors: Johansson Inger , Eliasson Erik , Norsten Carina , Ingelman-Sundberg Magnus
Published: July 2002

Acetone oxidation in rat liver microsomes was induced 5‐ or 8‐fold by the treatment of the animals with ethanol or acetone, respectively. The apparent K
m of the reaction was 0.9 mM, a value lower than the concentration reported for plasma acetone under starvation conditions. The major acetone metabolite was identified as acetol by GC‐MS. Acetone oxidation in microsomes was inhibited by typical P‐450 inhibitors as well as by compounds (e.g. imidazole) known to interact with the ethanol‐inducible P‐450 form. Antibodies against this P‐450 isozyme were inhibitory for the reaction in rabbit liver microsomes and this isozyme was the only one that showed acetone hydroxylation activity in reconstituted membranes. Imidazole inhibited the conversion of [14C]acetone into low‐M
r compounds (e.g. glucose) in vivo. It is suggested that the ethanol‐and acetone‐inducible P‐450 make use of acetone as an endogenous substrate in the utilization of the compound for, e.g. glucose production under conditions of starvation and diabetic ketoacidosis.
A fluorescence assay for monitoring and analyzing fusion of biological membrane vesicles in vitro
Authors: Stutzin Andres
Published: July 2002

A new technique has been developed to study fusion of biological membrane vesicles. Bovine chromaffin granule ghosts (CGG) were loaded with fluorescein isothiocyanate‐dextran (FITC‐dextran) at self‐quenching concentrations. Loaded ghosts were then made to fuse with empty CGG. Fusion was induced by synexin, a protein previously proposed to be involved in exocytosis. The fusion process was monitored by measuring the dequenching of the fluorescence. Dequenching occurred as FITC‐dextran was diluted into the increased volume due to fusion with empty ghosts. Spurious signals from leakage or breakage of vesicles were removed by including a specific anti‐fluorescein antibody in the reaction medium. This new technique may prove to be of more general use for studying membrane fusion processes in other systems.
Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine
Authors: Ancelin Marie L. , Vial Henri J.
Published: July 2002

In Plasmodium falciparum‐infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 ± 1.4 nmol phosphethanolamine/107 infected cells per h) was higher than choline kinase specific activity (1.9 ± 0.2 nmol phosphocholine/107 infected cells per h). The K
m of choline kinase for choline was 79 ± 20 μM, and ethanolamine was a weak competitive inhibitor of the reaction (K
i = 92 mM). Ethanolamine kinase had a K
m for ethanolamine of 188 ± 19 μM, and choline was a competitive inhibitor of ethanolamine kinase with a very high K
m of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D‐2‐amino‐1‐butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50°C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.
Formation of lipoxin A by granulocytes from eosinophilic donors
Authors: Serhan Charles N. , Hirsch Ulf , Palmblad Jan , Samuelsson Bengt
Published: July 2002

The formation of arachidonic acid‐derived lipoxygenase products was examined with human granulocytes obtained from eosinophilic donors. These eosinophil‐enriched leukocyte populations, challenged in vitro with the ionophore of divalent cations A23187, transformed both exogenous and endogenous sources of arachidonic acid to several lipoxygenase‐derived products, including 5(S),6(R),15(S)‐trihydroxy‐7,9, 13‐trans‐11‐cis‐eicosatetraenoic acid (lipoxin A). Lipoxin A was detected and characterized by high‐pressure liquid chromatography (HPLC), ultraviolet absorbance, and gas‐liquid chromatography‐mass spectroscopy. Neither lipoxin B nor 6(S)‐LXA was consistently detected in extracts from these incubations. The amounts of lipoxin A formed were proportional to the percentage of eosinophils present in the suspension. The results indicate that granulocytes from eosinophilic donors can generate lipoxin A.
Mast cell activation — a receptor‐independent mode of substance P action?
Authors: Repke H. , Bienert M.
Published: July 2002

Substance P is a representative of a group of amphiphilic neuropeptides which act as mast cell secretagogues. Our experiments with some new substance P derivatives suggest that these effects are dependent on two structural elements: (i) a hydrophobic chain which is not essentially a peptide, and (ii) a hydrophilic part with two positively charged amino acids. The mast cell triggering effect is unlikely to be mediated by a selective substance P receptor, but has strong similarities to the mode of action of polycations.
Generation of OH radical during enzymatic reduction of 9,10‐anthraquinone‐2‐sulphonate Can semiquinone decompose hydrogen peroxide?
Authors: Sushkov D.G. , Gritsan N.P. , Weiner L.M.
Published: July 2002

Spin‐trapping experiments have shown that 9, 10‐anthraquinone‐2‐sulphonate (AQS) can be reduced enzymatically with NADPH‐cytochrome P‐450 reductase (FP) producing OH radicals. It has been revealed for the first time that the radical anion of AQS cannot decompose H2O2 as indicated by flash‐photolysis data. From the experimental results obtained it follows that an enzyme system containing NADPH, AQS and FP produces both accumulation of H2O2 from molecular oxygen and reduction of Fe3+ to Fe2+ which decomposes H2O2 catalytically via a Fenton reaction.
The insulin receptor tyrosyl kinase phosphorylates holomeric forms of the guanine nucleotide regulatory proteins Gi and Go
Authors: O'Brien Richard M. , Houslay Miles D. , Milligan Graeme , Siddle Kenneth
Published: July 2002

An affinity purified human insulin receptor preparation was shown to phosphorylate the α‐ and β‐subunits of the guanine nucleotide‐regulatory proteins Gi and Go, derived from bovine brain. The presence of insulin stimulated the rate of their phosphorylation some 2‐fold. The presence of Gi and Go did not affect the degree of autophosphorylation of the β‐subunit of the insulin receptor. Under conditions known to cause the dissociation of Gi and Go into their constituent subunits then phosphorylation of Gi and Go by the insulin receptor was abolished. The α‐subunits of Gi and Go could be selectively phosphorylated by the insulin receptor tyrosyl‐kinase using appropriate concentrations of Mg2+ and GTP‐γ‐S.
Human immunoglobulin heavy‐chain multigene deletions in healthy individuals
Authors: Lefranc Marie-Paule , Lefranc Gérard
Published: July 2002

Extensive multigene deletions have been described in the human immunoglobulin heavy‐chain constant region genes, some of them encompassing perhaps more than 100 kilobases. These deletions have all been observed in healthy individuals although these individuals lacked several immunoglobulin class or subclasses, being either homozygous for one deletion or heterozygous for two different deletions. The high frequency of consanguinity in the Tunisian population accounts for the high frequency of individuals displaying one or the orther of these deletions in a homozygous state.
Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding α3‐subunits of rat brain Na+,K+‐ATPase
Authors: Hara Yukichi , Nikamoto Akira , Kojima Toshiyuki , Matsumoto Akiko , Nakao Makoto
Published: July 2002

The cDNAs encoding α3‐subunits of rat brain Na+,K+‐ATPase and the neomycin resistance gene were incorporated into BALB/c 3T3 cells by the co‐transfection method. Stably transformed cells were selected with 300 μg/ml of neomycin (G‐418) for 6 weeks. Northern blot analysis using the 3′‐non‐translated region of the cDNA as a probe revealed that the α3 mRNA appeared in transfected cells. Na+,K+‐ATPase activity of the transfected cells was twice that of wild‐type cells. Regarding ouabain sensitivity, the Na+,K+‐ATPase showed two K
i values for ouabain (8 × 10−8 and 4.5 × 10−5 M) in transfected cells while wild‐type cells displayed only the higher value. Ouabain sensitivity of Rb+ uptake also demonstrated two Ki values in the transfected cells (8 × 10−8 and 4.5 × 10−5 M) and a K
i in wild‐type cells of 4 × 10−5 M. It is concluded that α3 is a highly ouabain‐sensitive catalytic subunit of Na+,K+‐ATPase. It is also suggested that ouabain sensitivity is exclusively determined by the properties of the α‐subunit rather than the β‐subunit. This is the first report on the catalytic charateristics of the α3 isoform of Na+,K+‐ATPase.
Release of the nocturnal inhibitor, car☐yarabinitol‐1 ‐phosphate, from ribulose bisphosphate car☐ylase/oxygenase by rubisco activase
Authors: Robinson Simon P. , Portis Archie R.
Published: July 2002

Car☐yarabinitol‐1‐phosphate (CA1P) is a naturally occurring inhibitor of ribulose‐1,5‐bisphosphate car☐ylase/oxygenase (rubisco) which appears in the leaves of some plants during darkness and occupies the ribulose‐1,5‐bisphosphate binding site. The involvement of the rubisco activase protein in the regulation of rubisco activity by CA1P was investigated. Addition of ribulose bisphosphate (RuBP) or 6‐phosphogluconate partially reversed CA1P inhibition by displacing the inhibitor from rubisco. Rubisco activase also partially reversed the inhibition but rubisco activase and RuBP together almost completely reversed CA1P inhibition of rubisco. Rubisco activase did not metabolize CA1P. Inhibition of rubisco by car☐yarabinitol‐1,5‐bisphosphate was not reversed by rubisco activase in the presence or absence of RuBP. The data suggest that rubisco activase interacts with rubisco to alter the RuBP binding site, thereby increasing the dissociation rate of bound CA1P from the enzyme.
A novel pyrophosphate fructose‐6‐phosphate 1‐phosphotransferase from carrot roots Relation to PFK from the same source
Authors: Wong Joshua H. , Kang T. , Buchanan Bob B.
Published: July 2002

A novel type of pyrophosphate fructose‐6‐phosphate 1‐phosphotransferase (PFP) has been purified from carrot roots. The enzyme differs from its counterpart studied from other sources in subunit composition, response to regulatory metabolites and kinetic parameters. The properties of the enzyme are in accord with its function in a tissue that stores substantial quantities of sucrose. In describing the carrot root PFP, we have systematically compared its properties to those of phosphofructokinase from the same tissue. The results support the conclusion that, despite noteworthy similarities, the two enzymes are independent proteins, unlikely to undergo interconversion.
Inhibition of adenine nucleotide transport through the mitochondrial porin by a synthetic polyanion
Authors: Benz Roland , Wojtczak Lech , Bosch Waltraud , Brdiczka Dieter
Published: July 2002

The effect of a synthetic polyanion of Mr 10 000 (a copolymer of methacrylate, maleate and styrene in a 1:2:3 proportion) was studied on isolated rat liver mitochondria and on mitochondrial porin reconstituted into lipid bilayer membranes. Increasing concentrations of the polyanion inhibited the adenyl kinase located between both mitochrondrial membranes in a dose‐dependent fashion. Upon addition of the detergent digitonin in increasing concentrations the adenyl kinase activity was fully reversible. In reconstitution experiments with mitochondrial porin the polyanion increased the voltage dependence of the pore in such a way that the pore is switched into the closed state at much smaller voltages than in the absence of the polyanion. The asymmetric addition of the polyanion resulted in an asymmetric shift of the voltage‐dependence of the pore. If the voltage is negative at the cis‐side (the side of the addition of the polyanion) the pore closed rapidly whereas it was always open for potentials of opposite polarity. The results are discussed on the basis of a modification of the gate properties of the mitochondrial porin by the polyanion and by the assumption that the closed state of the pore is not permeable for nucleotides.
Detection of intercalation‐induced changes in DNA structure by reaction with diethyl pyrocarbonate or potassium permanganate Evidence against the induction of Hoogsteen base pairing by echinomycin
Authors: Jeppesen Claus , Nielsen Peter E.
Published: July 2002

Binding of the bis‐intercalators echinomycin and N,N′‐di(9‐acridinyl)spermidine or the mono‐intercalators 9‐aminoacridine and ethidium to DNA induces hypersensitivity of adenines towards reaction with diethyl pyrocarbonate. It is proposed that this hyperreactivity is due to the DNA helix unwinding and extension induced by intercalation, thereby exposing N7 in the major groove, and not as previously suggested to the formation of Hoogsteen base pairing. Hypersensitivity of thymines towards oxidation with permanganate is also induced upon binding of these drugs (especially the bis‐intercalators) to DNA. This thymine hyperreactivity is both sequence‐ and intercalator‐dependent, thereby indicating the potential of KMnO4 as a useful probe for analysing the structure of intercalator‐DNA complexes in solution.
Linolenic acid‐induced release of Mn, polypeptides and inactivation of oxygen evolution in photosystem II particles
Authors: Garstka Maciej , Kaniuga Zbigniew
Published: July 2002

The release of both Mn and polypeptides as well as the inactivation of O2 evolution in C18:3‐treated PS II particles was studied. An increase of incubation temperature above 20°C greatly accelerated depletion of both Mn and peptides and inactivation of O2 evolution. At 25°C about 30% of Mn was released whereas following incubation at 30°C release of 50% of Mn, complete depletion of both 10 and 18 kDa peptides as well as complete inactivation of O2 evolution were observed. Simultaneously, the release of 24 and both 33 and 43 kDa peptides was about 40 and 30%, respectively. At 25°C, C18:3 released only one Mn/reaction centre from PS II particles depleted of 18 and 24 kDa peptides, while two Mn/reaction centre were released from particles devoid of 18, 24 and 33 kDa peptides. These observations suggest that the 33 kDa peptide stabilizes preferentially two of the four Mn atoms of the oxygen‐evolving complex. The effect of C18:3 on Mn release from PS II particles is thus similar to that of heat and urea treatments.
Protochlorophyllide reductase: A flavoprotein?
Authors: Walker Caroline J. , Griffiths W.Trevor
Published: July 2002

Protochlorophyllide reductase (EC 1.6.99.1) catalyses the light‐dependent reduction of protochlorophyllide to chlorophyllide. Evidence has been obtained for the possible involvement of FAD in this reaction: (i) protochlorophyllide reductase is inhibited by quinacrine and trifluoperazine at micromolar levels consistent with their action as flavoprotein antagonists; (ii) preparations containing protochlorophyllide reductase can mediate cytochrome c reduction; (iii) in protochlorophyllide reductase‐rich etioplast membranes FAD co‐purifies with the enzyme.
Cloning and sequence analysis of human pituitary cDNA encoding the novel polypeptide 7B2
Authors: Martens Gerard J.M.
Published: July 2002

Application of a differential hybridization technique led to the isolation of a human pituitary cDNA clone encoding the complete structure of the polypeptide 7B2. This protein of unknown function, which is sorted to secretory granules, appears to be present selectively in neurons and endocrine cells. The polypeptide chain of human 7B2, preceded by a cleaved signal peptide, comprises 185 amino acids (a calculated M
r of 20 793). Interesting features of the highly‐conserved 7B2 structure include (i) a serine phosphorylation consensus sequence, (ii) the occurrence of three pairs of dibasic amino acids representing potential proteolytic cleavage sites and, in particular, (iii) the presence of three regions homologous to GTP‐binding domains giving 7B2 structural characteristics of a GTP‐binding protein.
Remarkable similarities between yeast and mammalian protein phosphatases
Authors: Cohen Philip , Schelling Donald L. , Stark Michael J.R.
Published: July 2002

Protein phosphatase activities in extracts of the yeast Saccharomyces cerevisiae showed remarkable similarities to the mammalian type 1, type 2A and type 2C enzymes. Similarities included their substrate specificities, including selectivity for the α‐ and β‐subunits of muscle phosphorylase kinase, sensitivity toadaic acid and to mammalian inhibitor 1 and inhibitor 2, and requirement for divalent cations. The results suggest that the function and regulation of these enzymes has been highly conserved during evolution and indicate that the improved procedure for identifying and quantitating protein phosphatases [(1989) FEBS Lett. 250, 000‐000] may be applicable to all eukaryotic cells.
Tissue specific distribution of calcyclin — 10.5 kDa Ca2+ ‐binding protein
Authors: Kuźnicki Jacek , Filipek Anna , Heimann Peter , Kaczmarek Leszek , Kamińska Boźena
Published: July 2002

Expression of calcyclin in different cell lines and mouse tissues was determined with polyclonal antibodies raised against calcyclin from Ehrlich ascites tumour (EAT) cells. The protein was detected in mouse skeletal and cardiac muscle, in lung, kidney and spleen, and was especially enriched in mouse smooth muscle as well as in rat fibroblasts. No positive immunological reaction was detected in mouse brain, liver and intestine and some tumourigenic cell lines. The level of calcyclin mRNA found in different cells and tissues corresponded well to the calcyclin level estimated by immunoblotting. The calcyclin‐like protein was purified from mouse stomach and appeared to be very similar to the EAT protein.
Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase
Authors: Van der Klei Ida J. , Lawson Catherine L. , Rozeboom Henriëtte , Dijkstra Bauke W. , Veenhuis Marten , Harder Wim , Hol Wim G.J.
Published: July 2002

Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X‐ray diffraction methods. The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 Å. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X‐ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.
A conserved cleavage‐site motif in chloroplast transit peptides
Authors: Gavel Ylva , von Heijne Gunnar
Published: July 2002

A collection of 32 stroma‐targeting chloroplast transit peptides with known cleavage sites have been analysed in terms of amino acid preferences in the vicinity of the processing site. A loosely conserved consensus motif (Val/Ile)‐X‐(Ala/Cys)↓Ala is found in the majority of the transit peptides. About 30% of the sequences have a perfect match to the consensus. When such a match is found, there is a 90% probability that it specifies the correct cleavage site.
Receptor‐active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99
Authors: Teneberg Susann , Willemsen Peter , de Graaf Frits K. , Karlsson Karl-Anders
Published: July 2002

Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin‐layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non‐acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor‐active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGcα‐3Galß4GlcßCer (NeuGc‐GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc‐GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N‐glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein‐linked sequences as well and thus explain the resistance of adult pigs to infection with E. coli K99.
Multiple control level governing H1o mRNA and protein accumulation
Authors: Khochbin Saadi , Gorka Claude , Lawrence Jean-Jacques
Published: July 2002

We have studied the variation of histone H1o and its coding mRNA during rat liver regeneration after partial hepatectomy. Our data showed that while H1o decreased when cell proliferation was initiated, H1o mRNA accumulated in a proliferantion‐dependent manner as did H3 mRNA. These results showed two interesting aspects of the regulation of H1o expression in vivo, confirming results we have obtained previously in vitro: first H1o mRNA accumulation is a proliferation‐dependent event; second, H1o protein accumulation may be uncoupled from that of its coding mRNA.
Binding of ATP to uncoupling protein of brown fat mitochondria as studied by means of spin‐labeled ATP derivatives
Authors: Jakobs P. , Braun A. , Jezek P. , Trommer W.E.
Published: July 2002

ATP derivatives spin‐labeled (SL) at C8, N6, C2′ or C3′ were employed in binding studies with the uncoupling protein of brown fat mitochondria. Substitution of the ribose strongly impaired binding, whereas labeling of the adenine moiety allowed for tight and functional complex formation. Detailed binding studies with C8‐SL‐ATP confirmed the known pH and Mg2+ dependence with a stoichiometry of one C8‐SL‐ATP bound per 66 kDa dimer. Corresponding studies of the uncoupling protein after modification with N‐ethylmaleimide or diazobenzene‐4‐sulfonic acid revealed distinct differences in their effects on nucleotide binding and gating.
Parental imprinting of the human H19 gene
Authors: Rachmilewitz Jacob , Goshen Ran , Ariel Ilana , Schneider Tamar , de Groot Nathan , Hochberg Abraham
Published: July 2002

It has only recently become clear that genetic imprinting plays an important role in human embryogenesis and in processes leading to the development of pediatric cancers and other human diseases. Using a unique human tissue, the androgenetic complete hydatidiform mole, we established that the maternally inherited allele of the imprinted H19 gene is expressed. Our results also show that the paternal allele of the human IGF‐II gene, a gene suspected to be parentally imprinted in humans, is expressed.
Regulatory light‐chain Cys‐55 sites on the two heads of myosin can come within 2Å of each other
Authors: Bower Susanne M. , Wang Yulai , Chantler Peter D.
Published: July 2002

The di‐thiol reagent, 5,5′‐dithiobis (2‐nitrobenzoic acid) is shown to induce disulfide bond formation between Mercenaria regulatory light‐chain Cys‐55 sites on either head of scallop hybrid myosin. This indicates that these two sites on opposite heads of myosin can come within 2Å of each other and this confirms a prediction based on earlier data [Chantler, Tao and Stafford (1991) Biophys. J. 59, 1242–1250]. Results demonstrate that myosin heads in solution show a considerable mutual freedom of movement which can be monitored by probes in the vicinity of regulatory light‐chain residue 55. Implications for light‐chain movement on the myosin head are discussed.
Purification of an endoproteinase that digests the wheat ‘Em’ protein in vitro, and determination of its cleavage sites
Authors: Taylor Richard M. , Cuming Andrew C.
Published: July 2002

Germinating wheat embryos contain two endoproteolytic activities which digest the prominent ‘Em’ polypeptide. These are easily assayed in clarified embryonic homogenates and are distinguishable by the pattern of their peptide products and by their different pH optima. One activity has a pH optimum of 4.0; the second activity is a cysteine endoproteinase with a preference for the ‘Em’ protein as its substrate. It is maximally active between pH 5.5 and 6 at 25°C. Analysis of the early cleavage products of the cysteine proteinase indicates scissile bonds between residues Glu32‐Ala33and Asn36‐Leu37in the ‘Em’ polypeptide. This endoproteinase has been purified and identified as a single polypeptide species of ca. 38,000 kDa.
Sucrose‐phosphate synthase phosphatase, a type 2A protein phosphatase, changes its sensitivity towards inhibition by inorganic phosphate in spinach leaves
Authors: Weiner Hendrik , Weiner Heike , Stitt Mark
Published: July 2002

The activity of a type 2A protein phosphatase from spinach leaves was monitored using phosphorylated sucrose‐phosphate synthase (SPS) as a substrate. After partial purification the overall activities of sucrose‐phosphate synthase phosphatase (SPS‐P) recovered from leaves harvested in the dark and in the light did not vary. However, SPS‐P preparations from darkened leaves were more strongly inhibited by inorganic phosphate and certain phosphorylated compounds than preparations from illuminated or mannose fed leaves. We conclude, that activation of SPS involves an interconversion of multiple forms of SPS‐P activity.
Probing the phalloidin binding site of actin
Authors: Faulstich Heinz , Zobeley Susanne , Heintz Daniela , Drewes Gerard
Published: July 2002

Phallotoxins form tight complexes with filamentous actin and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol‐capturing moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys‐374 next to the C‐terminus and Cys‐10, close to the N‐terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP‐G‐actin. Phalloidin bound to Cys‐374 stabilizes filaments against shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C‐terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F‐actin, however, the subsequent formation of a covalent linkage with Cys‐374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization of filaments by phalloidin linked to Cys‐374 a destabilizing effect was observed when phalloidin was attached to Cys‐10. It appears that phalloidin located close to the N‐terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxin is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phalloidin binding site of actin is distant from the N‐terminus, but close to the C‐terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C‐terminus.
Altered metabolism of glutathione in cells transformed by oncogenes which cause resistance to ionizing radiations
Authors: Vincenzini Maria Teresa , Marraccini Patrizia , Iantomasi Teresa , Favilli Fabio , Pacini Stefania , Ruggiero Marco
Published: July 2002

We measured glutathione (GSH) metabolism in normal NIH/3T3 fibroblasts, and in cells transformed by the oncogenes sis, erbB, src, ras, dbl, and raf.erbB,src,ras and raf, but not sis and dbltransformants, showed increased level of total and reduced GSH as compared with normal NIH/3T3 fibroblasts; oxidized GSH was elevated only in src‐ and ras‐transformed cells. Increased total GSH content was associated with decreased activity of the synthetic enzyme γ‐glutamylcysteine synthetase, and oxidized GSH level with increased activity of GSH reductase. These data suggest that GSH synthesis was selectively enhanced in cells transformed by specific oncogenes, with resulting down‐regulation of its synthetic enzyme; alterations of GSH metabolism appeared to be peculiar of transformation by specific oncogenes, and not trivial epiphenomena of neoplastic transformation. Oncogenic transformants that presented elevated level of GSH were also those reported to be resistant to antineoplastic drugs and ionizing radiations, thus confirming a possible link between altered GSH metabolism and resistance to antineoplastic treatment.
Enzymatic RNA synthesis with deoxynucleoside 5'‐O‐(1‐thiotriphosphates)
Authors: Gaur Rajesh K. , Krupp Guido
Published: July 2002

We have investigated the incorporation of 2'‐deoxynucleoside‐5'‐O‐(1‐thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [α‐S]dGTP, we obtained full‐length transcripts of pre‐tRNAphe and pre‐tRNAtyr using an appropriate mixture of 2'‐deoxynucleoside 5'‐O‐(1‐thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [α‐S]dTTP and [α‐S]dATP, were inserted specifically. However, [α‐S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative.
Cartilage oligomeric matrix protein (COMP) is an abundant component of tendon
Authors: DiCesare Paul , Hauser Nik , Lehman Daniel , Pasumarti Subhalakshmi , Paulsson Mats
Published: July 2002

An abundant matrix protein was purified under native conditions from adult bovine tendon and identified as cartilage oligomeric matrix protein (COMP) by immunochemical crossreaction, amino acid sequence identity of tryptic peptides derived from both N‐ and C‐terminal regions, and structure revealed by electron microscopy. Immunohistochemistry showed age‐dependent differences in distribution of COMP in tendon.
The 26S proteasome of the yeast Saccharomyces cerevisiae
Authors: Fischer Michael , Hilt Wolfgang , Richter-Ruoff Birgit , Gonen Hedva , Ciechanover Aaron , Wolf Dieter H.
Published: July 2002

Proteasomes are large multicatalytic proteinase complexes found in all eukaryotic organisms investigated so far. They have been shown to play a central role in cytosolic and nuclear proteolysis. According to their sedimentation coefficients two types of these particles can be distinguished: 20S proteasomes and 26S proteasomes. In contrast to 20S proteasomes, which were mainly characterized on the basis of their ability to cleave small chromogenic peptide substrates and certain proteins in an ATP‐independent manner, 26S proteasomes degrade ubiquitinylated proteins in an ATP‐dependent reaction. 20S proteasomes have been found in all eukaryotes from yeast to man. So far 26S proteasomes have only been discovered in higher eukaryotes. We now report the existence of the 26S proteasome in a lower eukaryote, the yeast Saccharomyces cerevisiae. Formation of the 26S proteasome could most effectively be induced in crude extracts of heat stressed yeast cells by incubation with ATP and Mg2+ ions. This treatment yielded a protein complex, which eluted from gel filtration columns at molecular masses higher than 1500 kDa. Besides chromogenic peptide substrates, this complex cleaves ubiquitinylated proteins in an ATP‐dependent fashion. In non‐denaturing‐PAGE, the purified 26S proteasome disintegrated and migrated as four protein bands. One of these bands could be identified as the 20S proteasome. On SDS‐PAGE, the 26S proteasome showed a complex pattern of subunit bands with molecular masses between 15 and 100 kDa. Further evidence for the 20S proteasome being the proteolytically active core of the 26S proteasome was obtained by following peptide cleaving activities in extracts of yeast strains carrying mutations in various subunits of the 20S proteasome.
The role of the ninth and tenth type III domains of human fibronectin in cell adhesion
Authors: Mardon Helen J. , Grant Kate E.
Published: July 2002

Fibronectins (FN) contain sites, in addition to the cell recognition site RGD in the tenth type III domain (FIII10), that are required for adhesive activity. The role of FIII10 and the adjacent FIII9 was analysed in functional cell adhesion assays recombinant FIII domains in which the domain boundaries were strictly conserved. FIII9 had no adhesive activity. FIII10, and FIII9 plus FIII10 had less activity than FN, whereas the activity of FIII9‐10 was similar to FN. We conclude that FIII9 acts synergistically with FIII10 in cell adhesion, and that this synergy is dependent upon the structural integrity of the FIII9‐10 pair of domains.
Structure and function of the corpus callosum in schizophrenia: What's the connection?
Authors: David AS , Minne C , Jones P , Harvey I , Ron MA
Published: July 2002

Tests of both structure and function of the corpus callosum have revealed abnormalities in schizophrenic patients. One such functional test employed lateralised Stroop stimuli presented tachistoscopically, to measure the transfer of interference and facilitation between the cerebral hemispheres. An attempt was made to relate indices of callosal transfer to clinical and demographic variables, including family history, as well as to indices of brain morphology. The latter included ventricle: brain ratio (VBR) measured by computed tomography (CT) scanning on 31 DSMIII schizophrenics, and the cross-sectional area of the corpus callosum from magnetic resonance imaging (MRI), obtained from 20 of these patients. VBR did not relate to functional measures; however, anterior callosal area correlated with indices of callosal connectivity. Patients with auditory hallucinations had smaller anterior callosal areas and tended to show less connectivity. The results show links between functional and structural measures of the corpus callosum, but their precise nature remains unclear.
Isopentenyl diphosphate isomerase deficiency in Synechocystis sp. strain PCC6803
Authors: Ershov Yuri , Gantt R.Raymond , Cunningham Francis X. , Gantt Elisabeth
Published: July 2002

Isopentenyl diphosphate isomerase (IPP isomerase) in many organisms and in plastids is central to isoprenoid synthesis and involves the conversion between IPP and dimethylallyl diphosphate (DMAPP). It is shown that Synechocystis PCC6803 is deficient in IPP isomerase activity, consistent with the absence in its genome of an obvious homologue for the enzyme. Incorporation of [1‐14C]IPP in cell extracts, primarily into C20, occurs only upon priming with DMAPP in Synechocystis PCC6803 and in Synechococcus PCC7942. Isoprenoid synthesis in these cyanobacteria does not appear to involve interconversion of IPP and DMAPP, raising the possibility that they are not within the plastid evolutionary lineage.
Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics
Authors: Celis Julio E , Kruhøffer Mogens , Gromova Irina , Frederiksen Casper , Østergaard Morten , Thykjaer Thomas , Gromov Pavel , Yu Jinsheng , Pálsdóttir Hildur , Magnusson Nils , Ørntoft Torben F
Published: July 2002

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.
High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper
Authors: Hoyne Peter A. , Cosgrove Leah J. , McKern Neil M. , Bentley John D. , Ivancic Neva , Elleman Thomas C. , Ward Colin W.
Published: July 2002

Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors. These soluble IRs have lowered affinity for insulin compared with full‐length IR, and exhibit linear Scatchard plots in contrast to the curvilinear plots obtained with full‐length IR, IR truncated at the C‐terminus of the transmembrane region and IR ectodomains fused to the self‐associating constant domains from Fc or λ immunoglobulins. In this report, we have fused the IR ectodomain to the 33 residue leucine zipper from the transcriptional activator GCN4 of Saccharomyces cerevisiae. This fusion protein binds insulin with high affinity in a manner comparable to native receptor. The respective dissociation constants were K
d1 8.2×10−11 M and K
d2 1.6×10−8 M for hIRedZip and K
d1 5.7×10−11 M and K
d2 6.3×10−9 M for membrane‐anchored, native receptor.
Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types
Authors: Zhang Xu Dong , Nguyen Tam , Thomas Wayne D , Sanders Jayne E , Hersey Peter
Published: July 2002

Resistance of normal cells to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis is believed to be mediated by expression of two decoy receptors. Here we show that the expression and localisation of TRAIL receptors (TRAIL‐Rs) vary between different cells and that resistance to TRAIL is mediated by different mechanisms. The decoy receptor, TRAIL‐R3, appeared important in protection of endothelial cells, whereas lack of surface death receptor expression and as yet unknown intracellular inhibitor(s) of apoptosis downstream of caspase‐3 may play a major role in protection of melanocytes and fibroblasts from TRAIL induced apoptosis, respectively. Differential subcellular location of decoy receptors may be an important determinant of their effectiveness in different types of normal cells.
Solution structure and membrane‐binding property of the N‐terminal tail domain of human annexin I
Authors: Yoon Mi-Kyung , Park Sang-Ho , Won Hyung-Sik , Na Doe-Sun , Lee Bong-Jin
Published: July 2002

The conformational preferences of AnxIN26, a peptide corresponding to residues 2–26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxIN26 adopts a mainly α‐helical conformation in membrane‐mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxIN26 showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp‐12 side‐chain, which was quite different between the two. The N‐terminal region of the AnxIN26 helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N‐terminal tail domain of human annexin I interacts with biological membranes in a partially calcium‐dependent manner.
Scar/WAVE is localised at the tips of protruding lamellipodia in living cells
Authors: Hahne Penelope , Sechi Antonio , Benesch Stefanie , Small J.Victor
Published: July 2002

Cell motility entails the extension of cytoplasmic processes, termed lamellipodia and filopodia. Extension is driven by actin polymerisation at the tips of these processes via molecular complexes that remain to be characterised. We show here that a green fluorescent protein (GFP) fusion of the Wiskott–Aldrich syndrome protein family member Scar1/WAVE1 is specifically recruited to the tips of lamellipodia in living B16F1 melanoma cells. Scar1–GFP was recruited only to protruding lamellipodia and was absent from filopodia. The localisation of Scar was facilitated by the finding that the formerly described inhibition of lamellipodia formation by ectopical expression of Scar, could be overcome by the treatment of cells with aluminium fluoride. These findings show that Scar is strategically located at sites of actin polymerisation specifically engaged in the protrusion of lamellipodia.
Identification of an interaction between SOX9 and HSP70
Authors: Marshall Owen J. , Harley Vincent R.
Published: July 2002

The campomelic dysplasia/autosomal sex reversal protein SOX9 is an important developmental transcription factor, required for correct bone and testis formation. Through in vitro and in vivo studies we have identified the heat shock protein HSP70 as an interacting partner for SOX9 in chondrocyte and testicular cell lines. HSP70 forms a ternary complex with DNA‐bound SOX9. The interaction between HSP70 and SOX9 is ATP‐independent and involves a highly conserved region of SOX9 hitherto of unknown function and the C‐terminal region of HSP70. Our results implicate HSP70–SOX9 interactions in the assembly of multi‐protein complexes during SOX9‐mediated transcription.
Caspases are reversibly inactivated by hydrogen peroxide
Authors: Borutaite Vilmante , Brown Guy C
Published: July 2002

Hydrogen peroxide (H2O2) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H2O2 inhibited the activity of recombinant caspase‐3 and caspase‐8, half‐inhibition occurring at about 17 μM H2O2. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H2O2 added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H2O2‐producing NADPH oxidase in macrophages also caused catalase‐sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H2O2.
α1‐β interaction in voltage‐gated cardiac L‐type calcium channels
Authors: Marquart Andrea , Flockerzi Veit
Published: July 2002

The β subunits of voltage‐gated calcium channels normalize current amplitude, kinetics and voltage dependence of these channels by interacting with the channel's pore forming subunit α1. By screening an epitope expression library of α1Ca fusion proteins, a β2a binding site of 22 amino acids was identified within the I–II cytoplasmic linker but not on other cytoplasmic sequences of α1Ca. This binding site overlaps by 14 amino acids with the conserved 18 amino acid peptide assumed to be essential for α1‐β interaction. The common 14 amino acid motif of α1Ca is sufficient to bind β2a, and in addition β1a, β3 and β4.
The novel estrogen receptor‐β subtype: potential role in the cell‐ and promoter‐specific actions of estrogens and anti‐estrogens
Authors: Kuiper George G.J.M , Gustafsson Jan-Åke
Published: July 2002

The recent discovery that an additional estrogen receptor (ER) subtype is present in various rat, mouse and human tissues has advanced our understanding of the mechanisms underlying estrogen signalling. The discovery of a second ER subtype (ERβ) suggests the existence of two previously unrecognised pathways of estrogen signalling: via the ERβ subtype in tissues exclusively expressing this subtype and via the formation of heterodimers in tissues expressing both ER subtypes. Various models have been suggested as explanations for the striking cell‐ and promoter‐specific effects of estrogens and anti‐estrogens, all on the basis of the assumption that only a single ER gene exists. This minireview describes several of these models and focuses on the potential role which the novel ERβ subtype might have in this regard.