bluuebunny/crossref_metadata_2025_split huggingface dataset:
cDNA clones for mouse parotid proline‐rich proteins
Authors: LAYFIELD Roy , BANNISTER Andrew J. , PIERCE Eric J. , MCDONALD Charles J. 
Published: March 2005

cDNA clones for mRNA sequences regulated by isoprenaline in mouse parotid glands were identified by differential colony hybridisation and all hybridised to a diagnostic proline‐rich protein (PRP) oligonucleotide. They were divided into two cross‐hybridisation groups, A and B, which were shown by hybrid‐selected translations to encode acidic PRP and basic PRP, respectively.The A‐type subgroup consisted of sequences homologous to the previously identified mouse PRP genes MP2 and MP3. The B‐type subgroup comprised clones for the previously identified cDNA pUMP125 (MP4) as well as other PRP sequences. Six of the B‐type clones contained a novel PRP cDNA (MP5) and these were sequenced. The composite MP5 cDNA was 897 nucleotides long and contained an open reading frame capable of encoding a 260‐residue‐long salivary PRP precursor (30% Pro, 19% Gln and 18% Gly), containing nine variant repeat units of consensus PGNQQGPPPQGGPQQ(GPP)R(PPQ). MP5 was 80% identical to the sequence of MP4 and had a high degree of similarity (60%) at its 3′‐untranslated region to rat salivary glutamate/glutamine‐rich protein (GRP) cDNA. Two MP5 clones contained a 273‐bp intron‐like insertion in the 3′ untranslated region, being derived, therefore, from incompletely spliced MP5 transcripts.Northern blotting showed that, although PRP mRNA species were induced by isoprenaline, a B‐type PRP mRNA was present in normal parotid glands. RNA dot‐blots probed with PRP‐genespecific oligonucleotides established that MP3, MP4 and MP5 PRP mRNA were all induced by isoprenaline.
An additional exon in the human vinculin gene specifically encodes meta‐vinculin‐specific difference peptide
Authors: KOTELIANSKY Victor E. , OGRYZKO Elena P. , ZHIDKOVA Natalia I. , WELLER Polly A. , CRITCHLEY David R. , VANCOMPERNOLLE Katia , VANDEKERCKHOVE Joel , STRASSER Peter , WAY Michael , GIMONA Mario , SMALL J. Victor 
Published: March 2005

We have analyzed the structure, origin and expression of the high‐molecular‐mass muscle‐specific variant of vinculin, called meta‐vinculin. The meta‐vinculin‐specific inserts from the human and avian molecules have been isolated and sequenced and the sequences confirmed via cloning of the corresponding cDNA. Comparison of the human, avian and determined porcine sequences revealed cross‐species identity in the C‐terminal half of the insert. Human and porcine meta‐vinculin were highly similar in the insert region, showing only five amino acid exchanges; avian meta‐vinculin showed 22 exchanges in the same region compared to human meta‐vinculin and exhibited, in addition, one extra amino acid, making 69 in all. Each insert was flanked by characteristic KWSSK motifs.Evidence for two vinculin mRNA species in human uterus smooth muscle was provided by reverse transcription combined with the polymerase chain reaction, as well as by ribonuclease‐mapping analysis of cDNA/mRNA hybrids. One of the mRNA species contained an additional 204‐nucleotide insert that precisely encoded the meta‐vinculin‐specific peptide.Sequence analysis of the appropriate portion of the human vinculin gene showed that the section coding for the meta‐vinculin‐specific insert is present as a discrete exon. Thus, meta‐vinculin and vinculin mRNA are generated by alternative splicing.
Intestinal absorption of β‐lactam antibiotics and oligopeptides
Authors: KRAMER Werner , GIRBIG Frank , GUTJAHR Ulrike , KOWALEWSKI Simone , ADAM Friedhelm , SCHIEBLER Werner 
Published: March 2005

The H+‐dependent uptake system responsible for the enteral absorption of oligopeptides and orally active β‐lactam antibiotics was functionally reconstituted into liposomes. Membrane proteins from rabbit small intestinal brush border membrane vesicles were solubilized with n‐octyl glucoside and incorporated into liposomes using a gel filtration method. At protein/lipid ratios of 1:10 and 1:40, the uptake of the orally active α‐amino‐cephalosporin, D‐cephalexin into proteoliposomes was stimulated by an inwardly directed H+ gradient and was protein‐dependent. In these proteoliposomes the binding protein for oligopeptides and β‐lactam antibiotics of Mr 127000 could be labeled by direct photoaffinity labeling with [3H]benzylpenicillin revealing an identical binding specificity as in the original brush border membrane vesicles.The uptake system for β‐lactam antibiotics and oligopeptides showed a remarkable stereospecificity; only D‐cephalexin was taken up by intact brush border membrane vesicles, whereas the L‐enantiomer was not taken up to a significant extent. This stereospecificity for uptake was also seen after reconstitution of solubilized brush border membrane proteins into liposomes demonstrating a functional reconstitution of the peptide transporter. Both enantiomers however, bound to the 127‐kDa binding protein as was shown by a decrease in the extent of photoaffinity labeling of the 127‐kDa protein in the presence of both enantiomers.After reconstitution of subfractions of brush border membrane proteins obtained by wheat germ lectin affinity chromatography into proteoliposomes, only liposomes containing the 127‐kDa binding protein showed a significant uptake of D‐cephalexin whereas the L‐enantiomer was not transported. The uptake rates for D‐cephalexin into proteoliposomes correlated with the content of 127‐kDa binding protein in these liposomes as was determined by specific photoaffinity labeling with [3H]benzylpenicillin. The purified 127‐kDa binding protein was also reconstituted into liposomes and its ability for specific binding of substrates as well as stereospecific uptake of cephalexin could be restored.These results indicate that the binding protein for oligopeptides and β‐lactam antibiotics of Mr 127000 mediates the stereospecific and H+‐dependent transport of orally active β‐lactam antibiotics across the enterocyte brush border membrane. We therefore suggest that this 127‐kDa binding protein is the intestinal peptide transport system (or a component thereof).
A comparative study of the temperature dependence of the oxygen‐binding properties of mammalian hemoglobins
Authors: COLETTA Massimo , CLEMENTI Maria Elisabetta , ASCENZI Paolo , PETRUZZELLI Raffaele , CONDÒ Saverio Giovanni , GIARDINA Bruno 
Published: March 2005

The effect of temperature on the oxygen‐binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3‐diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy].Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.
Control of plasminogen‐activator inhibitor type 2 gene expression in the differentiation of monocytic cells
Authors: ANTALIS T. M. , DICKINSON J. L. 
Published: March 2005

Plasminogen‐activator inhibitor type 2 (PAI‐2) is a potent and primary inhibitor of urokinase‐type plasminogen activator. Its production in monocytic cells is thought to play an important role in the control of localized proteolysis at sites of invasion as occurs in the control of inflammatory processes, tumor invasion and cellular differentiation. Therefore, we have investigated the mechanisms responsible for the regulation of PAI‐2 gene expression in differentiating monocytic cells using the human promyelocytic cell line, HL‐60, as a model. These cells are induced to differentiate to a macrophage‐like phenotype in response to phorbol ester [4‐phorbol‐12‐myristate 13‐acetate (PMA)]. The levels of PAI‐2 mRNA are barely detectable in undifferentiated cells, however, activation with PMA is associated with a rapid induction of PAI‐2 transcripts, reaching a maximum of 25‐fold in 4 h. Nuclear run on assays demonstrate that this induction is related primarily to an enhanced rate of gene transcription. Inhibition of de novo protein synthesis by cycloheximide increases PAI‐2 mRNA levels in both resting (sevenfold) and PMA‐treated cells (fivefold) after 4 h, but has no detectable effect on the rate of PAI‐2 gene transcription. The initial apparent half‐life of the induced PAI‐2 mRNA, determined by actinomycin‐d‐decay experiments, is very short, 32 min, suggesting rapid turnover. Furthermore, the PAI‐2 mRNA transcript is stabilized in the presence of cycloheximide, with a fourfold increase in the observed half‐life.The results demonstrate that PAI‐2 gene expression is regulated through post‐transcriptional mechanisms in undifferentiated cells, while both transcriptional and post‐transcriptional events govern the level of PAI‐2 transcripts in cells differentiated along the monocytic pathway. Destabilization of the PAI‐2 transcript may be associated with (A + U)‐rich sequences found in the 3′‐untranslated region of PAI‐2 mRNA. The short half life and rapid, strong induction of PAI‐2 point to an important, perhaps crucial, role in the differentiation of monocyte cells.
Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium
Authors: PLAGA Wulf , LOTTSPEICH Friedrich , OESTERHELT Dieter 
Published: March 2005

An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant.The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin‐diphosphate‐binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.
Identification and characterization of the murine cell surface receptor for the urokinase‐type plasminogen activator
Authors: SOLBERG Helene , LØBER Dorte , ERIKSEN Jens , PLOUG Michael , RØNNE Ebbe , BEHRENDT Niels , DANØ Keld , HØYER‐HANSEN Gunilla 
Published: March 2005

Cell‐binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase‐type plasminogen activator (u‐PA). In contrast to the human system, chemical cross‐linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand‐blotting analysis, two mouse u‐PA‐binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u‐PA receptor (u‐PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u‐PAR due to cross‐hybridization and pronounced sequence similarity with human u‐PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763–1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand‐blotting analysis. Binding of mouse u‐PA to its receptor showed species specificity in ligand‐blotting analysis, since mouse u‐PA did not bind to human u‐PAR and human u‐PA did not bind to mouse u‐PAR. The apparent Mr of mouse u‐PAR varied between different mouse cell lines and ranged over Mr 45000–60000. In four of the cell lines, mouse u‐PA bound to two mouse u‐PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u‐PA‐binding protein. In the monocyte macrophage cell line P388D.1, trypsin‐treatment of intact cells could remove only the large mouse u‐PAR variant (Mr 60000) indicating that only this type was a cell‐surface‐exposed molecule. The smaller mouse u‐PAR variant (Mr 45000), was deglycosylated by the enzyme endo‐β‐N‐acetylglucosaminidase H and is probably an intracellular precursor form carrying only high‐mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent Mr of about 30000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u‐PAR. Receptor‐bound mouse u‐PA could be released by phosphatidylinositol‐specific phospholipase C treatment, indicating that mouse u‐PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u‐PAR variant proteins by diisopropylfluorophosphate‐inactivated mouse u‐PA ‐ Sepharose affinity chromatography yielded two silver‐stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand‐blotting analysis.The present identification and characterization of mouse u‐PAR will facilitate experimental studies in vivo on the role of u‐PAR and u‐PA/u‐PAR interaction in normal and pathological conditions, including cancer invasion.
Concentration and regulation of cyclic nucleotides, cyclic‐nucleotide‐dependent protein kinases and one of their major substrates in human platelets
Authors: EIGENTHALER Martin , NOLTE Christine , HALBRÜGGE Maria , WALTER Ulrich 
Published: March 2005

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46‐kDa protein (vasodilator‐stimulated phosphoprotein, VASP) mediated by cAMP‐dependent protein kinase (PKA) and cGMP‐dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA‐ and PKG‐mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 μM cAMP and 3.1 μM catalytic subunit of PKA, which is equivalent to 6.2 μM cAMP‐binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 μM cGMP and 7.3 μM PKG monomer, equivalent to 14.6 μM cGMP‐binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 μM.Treatment of washed human platelets with 10 μM (or 10 nM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 μM (10 μM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46‐kDa protein) to the phosphorylated form (50‐kDa protein).Treatment of washed human platelets with 100 μM (or 1 μM) sodium nitroprusside elevated the platelet cGMP level to 4 μM (0.9 μM with 1 μM sodium nitroprusside) within 2 min, accompanied by a less‐rapid VASP phosphorylation of 45% (27% with 1 μM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increases in cAMP levels are required for activation of most of PKA, whereas even several‐fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA‐ and cGMP/PKG‐regulated VASP phosphorylation are components of an efficient and sensitive signal‐transduction pathway, most likely involved in the inhibition of platelet activation. In particular, the high concentration of the converter enzymes PKA and PKG suggest that rapidity in reaching steady‐state levels of phosphorylation in response to cAMP‐ or cGMP‐elevating agents is of considerable importance for the regulation of human platelets.
A single‐residue exchange gives human recombinant ββ alcohol dehydrogenase γγ isozyme properties
Authors: HÖÖG Jan‐Olov , EKLUND Hans , JÖRNVALL Hans 
Published: March 2005

Amino acid residue 48 in human alcohol dehydrogenase constitutes one of 21 residue differences between the common, adult‐type isozyme subunits β and γ. It is at the inner part of the substrate pocket and has been ascribed a role in hydrogen‐bond formation with both the substrate and coenzyme. In order to allow direct evaluation of its importance, Thr48 of a recombinant non‐acetylated β subunit was mutated to Ser (as in the γ subunit) or Ala (as in no native form, and not allowing side‐chain hydrogen bonds), and the proteins were expressed in Escherichia coli. The two non‐acetylated recombinant proteins, the β48T form and the mutant β48S, gave enzymatically active enzymes with indistinguishable specific activities towards ethanol, whereas the mutant β48A showed no enzymatic activity.The most striking differences between dimers with the β subunit and the β48S subunit (both non‐acetylated) were observed with cyclohexanol, hydroxysteroids, methanol and ethanol. With cyclohexanol, the Km was lowered from 11 mM to 280 μM, and the kcat/Km ratio, although still less than that for the γγ isozyme, was increased 80‐fold. Similarly, β48S could use 3β‐hydroxy‐5β‐androstan‐17‐one as substrate, like γγ, although again with a catalytic efficiency much less than that for the γγ isozyme. Furthermore, testosterone inhibited β48S to 50% at a concentration of 100 μM, whereas the ββ form was not inhibited. All these results show that residue 48 is responsible for a large part of the differences between the two isozymes ββ and γγ of human class‐I alcohol dehydrogenase. The form with the inactive β48A subunit was possible to purify by AMP‐Sepharose chromatography, suggesting the presence of a functional NAD‐binding site. The enzymatic measurements, demonstrating a transition from one isozyme activity to that characteristic of another, confirmed that a side‐chain hydroxyl in residue 48 is required for activity, and interpretation by computer modelling showed marked differences at the active site.
A simple procedure for tenascin purification
Authors: SAGINATI Marco , SIRI Annalisa , BALZA Enrica , PONASSI Marco , ZARDI Luciano 
Published: March 2005

Here we describe a two‐step procedure for purification of human tenascin from conditioned medium of the SK‐MEL‐28 human melanoma cell line.The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin–Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5–300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin‐sulfate‐containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3–4 mg tenascin which corresponds to a yield of about 40–50%.This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.
Apparent lack of N‐glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum
Authors: DIECKMANN‐SCHUPPERT Angela , BENDER Stephanie , ODENTHAL‐SCHNITTLER Maria , BAUSE Ernst , SCHWARZ Ralph T. 
Published: March 2005

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum, and the presence in the infected erythrocyte of the respective precursors.In in vitro cultures, P. falciparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein‐glycosylation reactions. Gel‐filtration analysis of sugar‐labeled malarial proteins before and after specific cleavage of N‐glycans or O‐glycans, respectively, revealed the majority of the protein‐bound sugar label to be incorporated into O‐glycans, but only little (7–12% of the glucosamine label) or no N‐glycans were found. Analysis of the nucleotide sugar and sugar‐phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP‐mannose was below the detection limit. The analysis of organic‐solvent extracts of sugar‐labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N‐glycosylation. Consistently, the amount of UDP‐N‐acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl‐transferase activity was not detectable in a lysate of P. falciparum, using exogenous glycosyl donors and acceptors.Our studies show that O‐glycosylation is the major form of protein glycosylation in intraerythrocytic P. falciparum, whereas there is little or no protein N‐glycosylation. A part of these studies has been published in abstract form [Dieckmann‐Schuppert, A., Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe‐Seyler 372, 645].