bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of a heat‐stable activator protein for ADP‐ribosylation factor‐dependent phospholipase D1
Authors: Akisue Toshihiro , Jinnai Hitoshi , Hitomi Tomohiro , Miwa Noriko , Yoshida Kimihisa , Nakamura Shun-ichi
Published: July 2002

A heat‐stable activator for ADP‐ribosylation factor (ARF)‐dependent phospholipase D (PLD) was purified to near homogeneity from rat kidney cytosol by a sequential column chromatography. The purified activator has a molecular mass of 23 kDa on SDS‐PAGE. Using a partially purified ARF‐dependent PLD from rat kidney, the activator synergistically stimulates PLD with ARF in time‐ and dose‐dependent manner. In the absence of ARF, the activator has little or no effect. The purified activator also stimulates PLD under several conditions including permeabilized cell system, suggesting that the activator is a physiologically relevant regulator of PLD.
Flow cytometric studies of nucleoside transport regulation in single chromaffin cells
Authors: Sen Raquel P , Delicado Esmerilda G , Alvarez Alberto , Brocklebank Anne M , Wiley James S , Miras-Portugal Mª Teresa
Published: July 2002

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5‐(SAENTA‐x8)‐fluorescein, is a highly specific inhibitor of the neural NBTI‐sensitive nucleoside transporter. 5‐(SAENTA‐x8)‐fluorescein inhibited adenosine transport and [3H]NBTI binding with a K
i of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5‐(SAENTA‐x8)‐fluorescein specifically interacted with the NBTI‐sensitive nucleoside transporters with high affinity (K
D=6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.
Recombinant protein kinase C‐γ phorbol binding domain upon microinjection blocked insulin‐induced maturation of Xenopus leavis oocytes
Authors: Pawelczyk Tadeusz , Matecki Andrzej , Dettlaff Anita
Published: July 2002

The second cysteine‐rich (Cys‐2) domain of rat brain PKC‐γ regulatory region C1 (92–173) was expressed in Escherichia coli cells and purified. NMR studies of Cys‐2 protein identified the phorbol and other phospholipid binding sites within this molecule (Xu, R.X., Pawelczyk, T., Xia, T‐H. and Brown, S.T. (1997) Biochemistry 37, 10709–10717). Here, we tested the ability of this domain to bind other proteins. Using an overlay assay we show that the Cys‐2 domain binds other proteins in Xenopus oocyte soluble fraction. Unlike the kinase activity, binding of Cys‐2 to other proteins was detected in the absence of added phospholipids. Microinjection of Cys‐2 protein into Xenopus leavis oocytes inhibited insulin‐induced but not progesterone‐induced maturation. the smallest dose that enhanced insulin‐induced maturation was 0.45×10−12 mol injected Cys‐2. These results demonstrate that the PKC‐γ Cys‐2 domain beside being the binding site for phorbol ester/DAG and phosphatidylserine binds also other proteins. The proteins that interact with Cys‐2 domain of PKC are essential for insulin‐induced maturation program in oocytes.
Mutations at residues Tyr771 and Tyr783 of phospholipase C‐γ1 have different effects on cell actin‐cytoskeleton organization and cell proliferation in CCL‐39 cells
Authors: Pei Zhen-Dong , Williamson John R
Published: July 2002

Currently a central question that remains unresolved is the potential importance of phospholipase C (PLC)‐γ1 in cell mitogenesis. In this study, we introduced wild‐type PLC‐γ1 plasmid and mutants Y771F and Y783F into CCL‐39 fibroblasts and investigated their effect on host cell functions. To our surprise, Y771F and Y783F plasmids appeared to have opposite effects on CCL‐39 cell actin‐cytoskeleton organization and cell proliferation. Y771F transfectants increased cell proliferation by two‐fold. Y783F transfectants showed much thicker actin filaments and decreased cell growth rate by 50%. These results suggest that PLC‐γ1 mutations have an essential impact on cell mitogenesis.
Alu elements in a Plasmodium vivax antigen gene
Authors: Dhar Arindam , Gupta Seema , Sharma Yagya D
Published: July 2002

Plasmodium vivax is a very common human malaria parasite but it is poorly characterized at the molecular level. Here, we describe the isolation and characterization of an antigen coding gene of P. vivax which contains Alu elements. This gene, called Pv‐Alu, is expressed during the erythrocytic phase of the parasite. The encoded 200 amino acid long polypeptide is highly hydrophobic, contains transmembrane domains, and is rich in leucine (19.4%), serine (15.9%), proline (15.4%) and phenylalanine (15.4%). The 5′‐untranslated region and part of the 3′‐end coding region of Pv‐Alu show significant homology to different Alu families. The presence of Alu elements in the coding region of a parasite antigen gene is significant from a functional and evolutionary viewpoint.
Protein synthesis in mitochondria isolated from the trypanosomatid protozoan Crithidia fasciculata
Authors: Tittawella Ivor
Published: July 2002

Evidence presented over the years in support of mitochondrial translation in trypanosomes, based largely on studies using differential inhibitors such as cycloheximide and chloramphenicol, remains controversial. I have studied endogenous mRNA‐dependent translation in a mitochondrial fraction isolated from the trypanosomatid protozoan Crithidia fasciculata. By using pancreatic ribonuclease to inactivate contaminating cytosolic activity, I show that these mitochondria can conduct protein synthesis in their own right. The mitochondrial translational products differed from cytosolic products as judged by SDS‐PAGE, and had sizes expected of some proteins encoded in the mitochondrial genome of C. fasciculata and other trypanosomatids. Some evidence is provided suggesting that the seat of translation might be the kinetoplast.
hUBC9 associates with MEKK1 and type I TNF‐α receptor and stimulates NFκB activity
Authors: Saltzman Alan , Searfoss George , Marcireau Christophe , Stone Maureen , Ressner Rose , Munro Robin , Franks Carol , D'Alonzo Jill , Tocque Bruno , Jaye Michael , Ivashchenko Yuri
Published: July 2002

hUBC9, an E2 ubiquitin conjugating enzyme, was identified by yeast two‐hybrid screening and coprecipitation studies to interact with MEKK1 and the type I TNF‐α receptor, respectively. Because both of these proteins regulate NFκB activity, the role of hUBC9 in modulating NFκB activity was investigated. Overexpression of hUBC9 in HeLa cells stimulated the activity of NFκB as determined by NFκB reporter and IL‐6 secretion assays. hUBC9 also synergized with MEKK1 to activate NFκB reporter activity. Thus, hUBC9 modulates NFκB activity which, at least in part, can be attributed to its interaction with MEKK1 and the type I TNF‐α receptor.
Pattern of methionine adenosyltransferase isoenzyme expression during rat liver regeneration after partial hepatectomy
Authors: Frago Laura M , Giménez América , Rodriguez Elı́as Nelson , Varela-Nieto Isabel
Published: July 2002

The present report investigates the pattern of expression of liver‐specific and extrahepatic methionine adenosyltransferase (MAT) isoenzymes in regenerating rat liver after partial hepatectomy. The results show that there is a switch in the expression of these isoenzymes that is coincident with maximal cell proliferation in the remaining liver lobes. Extrahepatic MAT levels increase about three times 4 h after hepatectomy, reaching a maximum 36 h later. This is accompanied by a rapid and transient increase in total MAT activity and levels of related metabolites S‐adenosyl‐l‐methionine and S‐adenosyl‐l‐homocysteine. Liver‐specific MAT levels are reversibly down‐regulated within 24–48 h post surgery. Our results indicate that MAT isoenzyme expression is tightly regulated during liver regeneration after two‐third hepatectomy. The implications of these observations for evaluation of the degree of liver regeneration are briefly discussed.
Quantitative structure activity relationships for the conversion of nitrobenzimidazolones and nitrobenzimidazoles by DT‐diaphorase: implications for the kinetic mechanism
Authors: Anusevicius Zilvinas , Soffers Ans E.M.F , Cénas Narimantas , Sarlauskas Jonas , Segura-Aguilar Juan , Rietjens Ivonne M.C.M
Published: July 2002

Quantitative structure activity relationships (QSARs) for the conversion of nitrobenzimidazolones and nitrobenzimidazoles by rat liver DT‐diaphorase (EC 1.6.99.2) are described. The parameter used for description of the QSARs is the energy of the lowest unoccupied molecular orbital (E(LUMO)) of the nitroaromatic compounds. Interestingly, correlations with E(LUMO) were observed for both the natural logarithm of k
cat, but also for the natural logarithm of k
cat/K
m. The minimal kinetic model in line with these QSARs is a ping‐pong mechanism that includes a substrate binding equilibrium in the second half reaction.
Thrombin induces the association of cyclic ADP‐ribose‐synthesizing CD38 with the platelet cytoskeleton
Authors: Torti Mauro , Festetics Enrico Tolnai , Bertoni Alessandra , Sinigaglia Fabiola , Balduini Cesare
Published: July 2002

The effect of platelet stimulation on the subcellular localization of CD38, a membrane glycoprotein that catalyses the synthesis of cyclic ADP‐ribose from β‐NAD+ was investigated. Treatment of human platelets with thrombin caused the association of about 40% of the total ADP‐ribosyl cyclase activity with the cytoskeleton, through the translocation of the CD38 molecule from the Triton X‐100‐soluble to the insoluble fraction. The interaction of CD38 with the cytoskeleton was a specific and reversible process, mediated by the binding to the actin‐rich filaments and was inhibited by treatment of platelets with cytochalasin D. This event was regulated by integrin αIIbβ3 and platelet aggregation as it was prevented by the inhibition of fibrinogen binding and was not observed in platelets from a patient affected by Glanzmann thrombasthenia. These results demonstrate that the subcellular localization of CD38 can be influenced by platelet stimulation with physiological agonists, and that membrane CD38 can interact with intracellular proteins.
DNA superstructural features and nucleosomal organization of the two centromeres of Kluyveromyces lactis chromosome 1 and Saccharomyces cerevisiae chromosome 61
Authors: Del Cornò Manuela , De Santis Pasquale , Sampaolese Beatrice , Savino Maria
Published: July 2002

Superstructural features of the Kluyveromyces lactis chromosome 1 (KlCEN1) and of the Saccharomyces cerevisiae chromosome 6 (SCEN6) centromeric DNAs were evaluated using a theoretical method, developed by our group, and experimentally measured by gel electrophoretic retardation. Both methods show that, in spite of the remarkable AT richness of the two centromeric sequences, their curvature is not very high. However the peculiar sequence features of the two centromeres allow to organize highly stable nucleosomes, with a free energy about that of the nucleosome formed on the 5S RNA gene. The good agreement between experimental and theoretical evaluation of nucleosome free energies as well as of their multiple positioning shows that in centromeres both DNA curvature and flexibility are relevant in determining nucleosomal features.
Selection of peptides that bind to plasminogen activator inhibitor 1 (PAI‐1) using random peptide phage‐display libraries
Authors: Gårdsvoll Henrik , van Zonneveld Anton-Jan , Holm Arne , Eldering Eric , van Meijer Marja , Danø Keld , Pannekoek Hans
Published: July 2002

Large random hexa‐ and decapenta‐peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa‐peptide library on immobilized plasminogen activator inhibitor 1 (PAI‐1) specifically selected a minor fraction of concatemers, indicating that binding to PAI‐1 requires an extended amino acid sequence. Accordingly, the decapenta‐peptide library exclusively yielded PAI‐1 binding peptides of 15 amino acid residues. None of these phage‐bound peptides prevented the interaction between PAI‐1 and its target serine protease urokinase (u‐PA). To isolate peptides that block the interaction between PAI‐1 and u‐PA, phages bound to immobilized PAI‐1 were eluted by incubation with u‐PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI‐1 or u‐PA.
Specific cleavage of γ catenin by caspases during apoptosis
Authors: Schmeiser Katja , Hammond Ester M , Roberts Sally , Grand Roger J.A
Published: July 2002

Caspase‐mediated proteolysis of cytoskeletal proteins during apoptosis appears to be commonplace. Enlarging on previous studies we have shown here that γ catenin, like β catenin, was degraded during cisplatin‐induced apoptosis, initially giving a major product of 75 kDa. This truncated protein could be co‐immunoprecipitated with α catenin. Addition of caspase inhibitors to cells in the presence of cisplatin appreciably reduced the proteolysis of γ catenin as well as the level of apoptosis. Only limited degradation of α catenin was observed even at very late times when over 90% of cells in the culture were apoptotic. Immunohistochemical staining showed that during apoptosis there was a relocation of α, β and γ catenin from the periphery of the cell to the cytoplasm, at the same time as other morphological changes commonly associated with apoptosis occurred. Interestingly, the changes in localisation of the catenins preceded proteolysis by several hours. In the presence of cisplatin and caspase inhibitor no change in distribution of catenins was observed, suggesting that re‐localisation requires caspase activity but not necessarily directed against β and γ catenins.
Pervanadate‐triggered MAP kinase activation and cell proliferation are not sensitive to PD 98059
Authors: Krady Marie-Marthe , Malviya Anant N. , Dupont Jean-Luc
Published: July 2002

A tight and stable complex with corresponding protein kinases and phosphatases establishes coupling between activators and inactivators. One such example is emerging from the studies of the Ras‐dependent MAP kinase cascade signaling pathway. Pervanadate, a potent inhibitor of protein tyrosine phosphatase, stimulates MAP kinase and elicits cell proliferation in cultured mouse fibroblasts which is insensitive to PD 98059, the major inhibitor of upstream MEK, whereas serum‐ or TPA‐ triggered proliferation is sensitive to PD 98059. It is suggested that imbalanced coordination between protein kinase and protein phosphatase determines the cellular responses such as cell proliferation. The PD 98059‐insensitive cell proliferation upon protein tyrosine phosphatase inhibition is attributed to a MEK bypass pathway.
Stimulation by anaphylatoxin C5a of glycogen phosphorylase in rat hepatocytes via prostanoid release from hepatic stellate cells but not sinusoidal endothelial cells
Authors: Schieferdecker Henrike L , Pestel Sabine , Rothermel Ellen , Püschel Gerhard P , Götze Otto , Jungermann Kurt
Published: July 2002

In the perfused rat liver, the anaphylatoxin C5a has been shown to enhance glucose output. Since hepatocytes lack C5a receptor mRNA, the metabolic effect of C5a must be elicited indirectly via C5a receptor expressing non‐parenchymal liver cells. Kupffer cells were found to be able to mediate the C5a action via release of prostanoids. However, elimination of the Kupffer cells by pretreatment of the animals with gadolinium chloride reduced the metabolic effect of C5a to only about 40%. Therefore, it was investigated whether not only Kupffer cells but in addition also hepatic stellate cells or sinusoidal endothelial cells released prostanoids in response to C5a. In isolated hepatic stellate cells but not in sinusoidal endothelial cells, recombinant rat C5a induced a time‐ and dose‐dependent release of thromboxane B2 and prostaglandins D2, E2 and F2α. The rate of prostanoid release was maximal within the first two minutes and then declined again. C5a‐induced prostanoid release from hepatic stellate cells was smaller than that from Kupffer cells and it differed in the prostanoid ratios (PGE2/PGD2/PGF2α/TXB2=1:1:0.1:0.6 and 1:4:1:3, respectively). RrC5a activated hepatocellular glycogen phosphorylase via prostanoid release in cocultures of hepatocytes with hepatic stellate cells but not with sinusoidal endothelial cells. Thus, the part of the rrC5a‐induced glucose output in the perfused rat liver, which was not abrogated by elimination of the Kupffer cells with gadolinium chloride, most likely was mediated by prostanoids released from hepatic stellate cells.
Different response to inflammation of the multiple mRNAs of rat N‐acetylglucosaminyltransferase I with variable 5′‐untranslated sequences1
Authors: Fukada Takashi , Kioka Noriyuki , Nishiu Jun , Sakata Shohei , Hiroshi Sakai  , Yamada Masayasu , Komano Tohru
Published: July 2002

We found that there are at least five subclasses of N‐acetylglucosaminyltransferase I (GnT‐I; EC 2.4.1.101) mRNA with different 5′‐untranslated regions in rat brain. These five subclasses were also expressed in many tissues with distinct tissue‐specific patterns. Moreover, they were regulated differently in response to acute‐phase inflammation. The expression of the most abundant subclass of GnT‐I mRNA in rat liver decreased 2.5‐fold in response to inflammation, concomitantly with a significant decrease in the total amount of GnT‐I mRNA. In contrast, one of the minor subclasses of GnT‐I mRNA was induced 10‐fold by inflammation.
Properties of a mutant form of the prokaryotic enhancer binding protein, NTRC, which hydrolyses ATP in the absence of effectors
Authors: Widdick David , Farez-Vidal Esther , Austin Sara , Dixon Ray
Published: July 2002

The mutation S170A in the proposed nucleotide binding site of the transcriptional activator protein NTRC abolishes its ability to catalyse open promoter complex formation by the σN‐RNA polymerase holoenzyme. NTRCS170A has significant ATPase activity, which, in contrast to the wild‐type protein, is unaffected by phosphorylation or binding to enhancer sites on DNA. The mutant protein appears to oligomerise normally on DNA in response to phosphorylation but the ATPase activity is apparently not responsive to changes in oligomerisation state. The defect in transcriptional activation is discussed in relation to mutations in other σN‐dependent activators.
In vitro assembly of a β2 cytochrome b559‐like complex from the chemically synthesised β‐subunit encoded by the Synechocystis sp. 6803 psbF gene
Authors: Francke Christof , Loyal Rosemarie , Ohad Itzhak , Haehnel Wolfgang
Published: July 2002

The α‐ and β‐subunits of cytochrome b
559 encoded by the psbE and psbF gene, respectively, are essential components of photosystem II. The exact structure of this cytochrome is not yet known. The β‐subunit of the Synechocystis sp. 6803 cytochrome b
559 complex was synthesised by means of solid‐phase peptide synthesis. Under reducing conditions, two β‐peptide molecules could be assembled specifically with one haem to form a β2 cytochrome b
559‐like complex. The spectral properties and the midpoint redox potential (48±5 mV) of the in vitro assembled β2 cytochrome are nearly identical to those of the low potential form of the native cytochrome b
559.
Cloning of a bovine orphan transporter and its short splicing variant
Authors: Sakata Kazuko , Shimada Shoichi , Yamashita Toshihide , Inoue Kiyoshi , Tohyama Masaya
Published: July 2002

We have isolated a cDNA (bv7‐3) encoding a member of the Na+,Cl−‐dependent transporter family and its short splicing variant (bv7‐3s) by screening a bovine retina cDNA library. Sequence analysis revealed that bv7‐3 encodes a protein of 729 amino acids and is a bovine homologue of the rat orphan transporter v7‐3‐2. bv7‐3s contains 265 amino acids, sharing 252 N‐terminal amino acids with bv7‐3. Both mRNAs for bv7‐3 and bv7‐3s were detected in nervous system by Northern blot analysis. In immunofluorescence analysis in transfected HEK 293T cells, myc‐tagged bv7‐3 was mainly detected on the plasma membrane, whereas myc‐tagged bv7‐3s showed a pattern of intracellular membrane staining.
Cell‐free immunology: construction and in vitro expression of a PCR‐based library encoding a single‐chain antibody repertoire
Authors: Makeyev Eugeny V. , Kolb Vyacheslav A. , Spirin Alexander S.
Published: July 2002

A novel cloning‐independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single‐chain antibody repertoire and express it directly in a cell‐free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.
Cytosolic phospholipase A2 in rat decidual cells: evidence for its role in decidualization
Authors: Kurusu S. , Endo M. , Madarame H. , Kawaminami M. , Hashimoto I.
Published: July 2002

We investigated the existence and possible role of cytosolic phospholipase A2 (cPLA2) in rat decidualized uteri. PLA2 activity in the cytosol of a decidualized uterine horn, induced by intraluminal oil infusion, was significantly higher than that in contralateral intact horn. The activity was almost completely depressed by cPLA2 inhibitors including arachidonyl trifluoromethyl ketone (ATK). The immunoreactive signals for cPLA2 were intense in decidua and glandular epithelial cells. In vivo administration of ATK (0.1–100 μg) caused a dose‐dependent inhibition of decidualization. These results show the presence of cPLA2 and its probable implication in decidualization in rat uterus.
Characterization of phosphotyrosine containing proteins at the cholinergic synapse
Authors: Balasubramanian Sudha , Huganir Richard L.
Published: July 2002

Tyrosine phosphorylation has been associated with several aspects of the regulation of cholinergic synaptic function, including nicotinic acetylcholine receptor (AChR) desensitization as well as the synthesis and clustering of synaptic components. While some progress has been made in elucidating the molecular events initiating such signals, the downstream targets of these tyrosine kinase pathways have yet to be characterized. In this paper we have used molecular cloning techniques to identify proteins which are tyrosine phosphorylated at the cholinergic synapse. Phosphotyrosine containing proteins (PYCPs) were isolated from the electric organ of Torpedo californica by anti‐phosphotyrosine immunoaffinity chromatography. Peptide sequencing and expression cloning then identified the isolated proteins. The proteins identified included heat shock protein 90, type III intermediate filament from Torpedo electric organ, α‐fodrin, β‐tubulin, actin and rapsyn. These tyrosine phosphorylated proteins may play a role in the regulation of synaptic function by tyrosine kinases.
Investigation of the interaction of the water oxidising manganese complex of photosystem II with the aqueous solvent environment
Authors: Evans Michael C.W , Gourovskaya Kira , Nugent Jonathan H.A
Published: July 2002

Interaction of the water oxidising manganese complex of photosystem II with the aqueous environment has been investigated using electron paramagnetic resonance spectroscopy and electron spin echo envelope modulation spectroscopy to detect interaction of [2H]methanol with the complex in the S2 state. The experiments show that the classical S2 multiline signal is associated with a manganese environment which is not exposed to the aqueous medium. An electron paramagnetic resonance spectroscopy signal, also induced by 200 K illumination, showing 2H modulation by methanol in the medium and a modified multiline electron paramagnetic resonance spectroscopy signal formed in parallel to it, are suggested to be associated with a second manganese environment exposed to the medium.
Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling
Authors: Hadad Nurit , Martin Cecile , Ashley Richard H. , Shoshan-Barmatz Varda
Published: July 2002

Two high M
r protein bands (440 and 420 kDa) in sheep brain microsomal membranes were labeled with the photoaffinity ATP analog, O‐(4‐benzoyl)benzoyl adenosine 5′‐triphosphate (Bz2ATP). The 420 kDa band is labeled by [α‐32P]‐Bz2ATP with about 1000‐fold higher affinity than the 440 kDa band. The heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and cross‐reactivity with anti‐dynein antibodies. The 440 kDa protein is immunologically identified as the type‐2 RyR. Bz2ATP binding is obtained in the absence of divalent cations. Bz2ATP and ATP increased the binding of ryanodine to its receptor up to 3‐fold, and increased the binding affinity up to 6‐fold. Other nucleotides stimulate ryanodine binding with decreasing effectiveness: Bz2ATP>ATP>ADP>AMP>AMP‐PNP>GTP>cAMP. With respect to nucleotide specificity, this binding site is similar to the skeletal muscle RyR (type 1). However, the brain RyR may have additional one or more sites with lower affinity with inhibitory effect on ryanodine binding. These results suggest that the major RyR isoform in sheep brain corresponds to the type‐2 isoform, and that modulation of ryanodine binding by ATP involves its binding to the RyR protein. The association of dynein with brain microsomal membranes may reflect a linkage of RyR to the cytoskeleton.
Insertional gene fusion technology
Authors: Doi Nobuhide , Yanagawa Hiroshi
Published: July 2002

The classical ‘end to end’ gene fusion technique has widely been used for monitoring gene expression, biological screening and purification of recombinant proteins. Recent progress with the ‘insertional’ gene fusion approach, on the other hand, has demonstrated that this technique can be utilized for membrane protein topology analysis, display of randomized protein libraries and design of biosensor proteins. In this review, we describe examples of insertional gene fusion and compare the old and new gene fusion techniques.
Inhibition of cell proliferation by lithium is associated with interference in cdc2 activation
Authors: Smits Veronique A.J , Essers Marieke A.G , Loomans Dominique S.E , Klompmaker Rob , Rijksen Gert , Medema René H
Published: July 2002

Lithium can interfere with embryonal development in a variety of organisms. We investigated the effect of lithium on the proliferation of early embryonal cells. [3H]Thymidine incorporation of non‐committed mouse P19 embryonal carcinoma cells was inhibited by lithium treatment. Similar effects were seen in a variety of other cells. This growth inhibition occurred in the G2 phase, since cells accumulated with a 4N DNA content, but the appearance of mitotic cells was blocked. Lithium could also prevent the activation of cdc2, thereby inhibiting cyclin B/cdc2 kinase activity. These data indicate that lithium might disturb embryonal development through interference in embryonal cell cycle regulation.
Cloning and characterization of a rabbit ortholog of human Gα16 and mouse Gα15
Authors: Feild John A. , Foley James J. , Testa Tania T. , Nuthulaganti Parvathi , Ellis Catherine , Sarau Henry M. , Ames Robert S.
Published: July 2002

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G‐protein α subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Gα16 and mouse Gα15, respectively. The rabbit Gα subunit cDNA was subcloned into a mammalian expression vector and transiently co‐transfected into HEK‐293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G α subunit behaved similarly to Gα15 or Gα16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit Gα subunit appears to be the ortholog of human Gα16 and mouse Gα15.
Identification of residues involved in binding of IL5 to βcom using βIL3 and βcom chimeras
Authors: Czabotar Peter E , Holland John , Sanderson Colin J
Published: July 2002

In mice there are two forms of the beta chain used in the IL3 receptor system, βcom and βIL3. βcom is used by the IL3, IL5 and GM‐CSF receptors whereas βIL3 is only used in the IL3 receptor. In this work an assay was developed to identify residues of βIL3 that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the βIL3 B′‐C′ loop were replaced with βcom sequence a form of βIL3 was produced that was able to respond to IL5. This region is also responsible for IL3 binding to βIL3 in the absence of alpha chain. It is therefore an important structural motif of βcom and βIL3 responsible for both ligand interaction and specificity.
Determination of the binding rate constants of stigmatellin and UHDBT to bovine cytochrome bc1 complex by cytochrome c1 oxidation
Authors: Zhang L , Snyder C , Trumpower B.L , Yu L , Yu C.A
Published: July 2002

Based on the high electron transfer rate between the [2Fe‐2S] cluster and heme c
1 and the elevation of the redox midpoint potential of iron sulfur protein (ISP) upon binding of certain Qo inhibitors, the binding rate constants of stigmatellin and UHDBT to the cytochrome bc
1 complex were determined using a stopped‐flow rapid scanning technique. Assuming that the intramolecular electron transfer from ISP to cytochrome c
1 is much faster than the binding of inhibitors, the rate of the inhibitor binding can be determined by the rate of cytochrome c
1 oxidation. The binding rate constants were calculated to be 1.0×105 and 2.3×105 M−1 s−1 at pH 7.5 for stigmatellin and UHDBT, respectively. The binding rate constant of UHDBT is pH dependent and that of stigmatellin is not.
Meprin mRNA in rat intestine during normal and glucocorticoid‐induced maturation: divergent patterns of expression of α and β subunits
Authors: Henning Susan J. , Oesterreicher Thomas J. , Osterholm Doreen E. , Lottaz Daniel , Hahn Dagmar , Sterchi Erwin E.
Published: July 2002

Meprin is a zinc‐metalloendopeptidase expressed in intestinal epithelial cells. In rat jejunum collected from postnatal day 4 (P4) through P25 meprins α mRNA exhibited uniform levels for the first three postnatal weeks and then declined, whereas meprin β mRNA showed a biphasic pattern with high levels in the first postnatal week followed by low levels from P7 through P19 and then a marked rise at P22 and P25. Dexamethasone treatment beginning at P10 had no significant effect on levels of meprins α mRNA, whereas this treatment caused a precocious increase in expression of meprin β mRNA. These divergent patterns of expression of meprins α and β mRNA suggest distinct roles for the two subunits during the suckling and weaning phases of rodent intestinal development.
Identification of a hydrogen bond in the Phe M197→Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X‐ray crystallography and FTIR spectroscopy1
Authors: Kuglstatter Andreas , Hellwig Petra , Fritzsch Günter , Wachtveitl Josef , Oesterhelt Dieter , Mäntele Werner , Michel Hartmut
Published: July 2002

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X‐ray structure analysis of the Phe M197→Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 Å resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875–12886, 1993] between the Tyr M197 hydroxy group and one of the 2a‐acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the π‐conjugated 9‐keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.
The role of phosphatidylinositide‐3‐kinase in basal mitogen‐activated protein kinase activity and cell survival
Authors: Versteeg Henri H , Evertzen Maaike W.A , van Deventer Sander J.H , Peppelenbosch Maikel P
Published: July 2002

Phosphatidylinositide‐3‐OH‐kinase (PI 3‐kinase) is an upstream activator of p42/p44 mitogen‐activated protein kinase (MAPK), but the role of PI 3‐kinase‐dependent MAPK remains obscure. Here we demonstrate that in a variety of different cell types, PI 3‐kinase inhibition results in an inhibition of MAPK in unstimulated cells but does not interfere with growth factor‐, or TPA‐induced MAPK activity. Furthermore, inhibition of either PI 3‐kinase or MEK/MAPK results in cell death in serum‐starved cells. We concluded that basal, but not induced MAPK activity is mediated by PI 3‐kinase and that this PI 3‐kinase‐mediated MEK/MAPK activity is essential for cell survival in quiescent cells.
The operation failed, but the patient survived. Varying assessments of the Soviet Union's last anti-alcohol campaign1
Authors: Reitan Therese C.
Published: July 2002

From the mid 1980s mortality levels have fluctuated greatly in the former Soviet Union. After dropping substantially during the late 1980s, mortality rose to unprecedented levels during the early 1990s. The sharp fluctuations in mortality are commonly linked to variations in alcohol consumption in connection with the anti-alcohol campaign launched in 1985. This large-scale natural alcohol policy experiment has produced very mixed appraisal and this article provides a systematic review of the wide variety of judgments, focusing on goals, implementation, and effects on life expectancy, alcohol consumption, mortality, crime, etc. Deviant evaluations are in part ascribable to a general schism between narrowly focused epidemiological perspectives on public health interventions and broader social science approaches to political reform.
Phosphorylation of the conserved C‐terminal domain of ribosomal P‐proteins impairs the mode of interaction with plant toxins
Authors: Horbowicz‐Drożdżal Patrycja , Kamel Karol , Kmiecik Sebastian , Borkiewicz Lidia , Tumer Nilgun E. , Shaw Pang‐Chui , Tchórzewski Marek , Grela Przemysław
Published: July 2021

The ribosome is subjected to post‐translational modifications, including phosphorylation, that affect its biological activity. Among ribosomal elements, the P‐proteins undergo phosphorylation within the C terminus, the element which interacts with trGTPases or ribosome‐inactivating proteins (RIPs); however, the role of phosphorylation has never been elucidated. Here, we probed the function of phosphorylation on the interaction of P‐proteins with RIPs using the ribosomal P1‐P2 dimer. We determined the kinetic parameters of the interaction with the toxins using biolayer interferometry and microscale thermophoresis. The results present the first mechanistic insight into the function of P‐protein phosphorylation, showing that introduction of a negative charge into the C terminus of P1‐P2 proteins promotes α‐helix formation and decreases the affinity of the P‐proteins for the RIPs.
FANCD2 limits acetaldehyde‐induced genomic instability during DNA replication in esophageal keratinocytes
Authors: Peake Jasmine D. , Noguchi Chiaki , Lin Baicheng , Theriault Amber , O'Connor Margaret , Sheth Shivani , Tanaka Koji , Nakagawa Hiroshi , Noguchi Eishi
Published: July 2021

Individuals with Fanconi anemia (FA), a rare genetic bone marrow failure syndrome, have an increased risk of young‐onset head and neck squamous cell carcinomas (SCCs) and esophageal SCC. The FA DNA repair pathway is activated upon DNA damage induced by acetaldehyde, a chief alcohol metabolite and one of the major carcinogens in humans. However, the molecular basis of acetaldehyde‐induced genomic instability in SCCs of the head and neck and of the esophagus in FA remains elusive. Here, we report the effects of acetaldehyde on replication stress response in esophageal epithelial cells (keratinocytes). Acetaldehyde‐exposed esophageal keratinocytes displayed accumulation of DNA damage foci consisting of 53BP1 and BRCA1. At physiologically relevant concentrations, acetaldehyde activated the ATR‐Chk1 pathway, leading to S‐ and G2/M‐phase delay with accumulation of the FA complementation group D2 protein (FANCD2) at the sites of DNA synthesis, suggesting that acetaldehyde impedes replication fork progression. Consistently, depletion of the replication fork protection protein Timeless led to elevated DNA damage upon acetaldehyde exposure. Furthermore, FANCD2 depletion exacerbated replication abnormalities, elevated DNA damage, and led to apoptotic cell death, indicating that FANCD2 prevents acetaldehyde‐induced genomic instability in esophageal keratinocytes. These observations contribute to our understanding of the mechanisms that drive genomic instability in FA patients and alcohol‐related carcinogenesis, thereby providing a translational implication in the development of more effective therapies for SCCs.
Insights into the ligand binding specificity of SREC‐II (scavenger receptor expressed by endothelial cells)
Authors: Wicker‐Planquart Catherine , Tacnet‐Delorme Pascale , Preisser Laurence , Dufour Samy , Delneste Yves , Housset Dominique , Frachet Philippe , Thielens Nicole M
Published: July 2021

SREC‐II (scavenger receptor expressed by endothelial cells II) is a membrane protein encoded by the SCARF2 gene, with high homology to class F scavenger receptor SR‐F1, but no known scavenging function. We produced the extracellular domain of SREC‐II in a recombinant form and investigated its capacity to interact with common scavenger receptor ligands, including acetylated low‐density lipoprotein (AcLDL) and maleylated or acetylated BSA (MalBSA or AcBSA). Whereas no binding was observed for AcLDL, SREC‐II ectodomain interacted strongly with MalBSA and bound with high affinity to AcBSA, a property shared with the SR‐F1 ectodomain. SREC‐II ectodomain also interacted with two SR‐F1‐specific ligands, complement C1q and calreticulin, with affinities in the 100 nm range. We proceeded to generate a stable CHO cell line overexpressing full‐length SREC‐II; binding of MalBSA to these cells was significantly increased compared with nontransfected CHO cells. In contrast, no increase in binding could be detected for C1q and calreticulin. We show for the first time that SREC‐II has the capacity to interact with the common scavenger receptor ligand MalBSA. In addition, our data highlight similarities and differences in the ligand binding properties of SREC‐II in soluble form and at the cell surface, and show that endogenous protein ligands of the ectodomain of SREC‐II, such as C1q and calreticulin, are shared with the corresponding domain of SR‐F1.
Retirement of a longtime leader triggers a soul‐searching process
Authors: Enos Gary
Published: July 2021

Any planned departure of an organization's top administrator generates a healthy amount of anxiety among staff, and often the community served as well. When the exiting leader has been at the helm for decades and has shepherded a successful organizational transformation, the level of concern about the future can rise exponentially.
Meeting: Make OUD medications available in jails and prisons
Authors: Knopf Alison
Published: July 2021

In Medications for Opioid Use Disorder in Jails and Prisons: Moving Toward Universal Access, a report summarizing a meeting April 20 led by the Johns Hopkins Bloomberg School of Public Health and updated last week, medical and public health experts from across the country call for making methadone and buprenorphine universally available in jails and prisons.
Thermal Properties of Laser‐Fabricated Copper–Carbon Composite Films on Polyimide Substrate
Authors: Yao Yu , Guo Wei , Zhou Xingwen , Peng Peng
Published: July 2021

The growing demand for highly efficient heat management in flexible electronics requires fabrication of highly conductive flexible films. Herein, Cu–C film on a polyimide (PI) substrate is fabricated to form a layered Cu–C/PI composite conductive film using direct laser writing. Its oxidation resistance is significantly improved compared with the written Cu film due to the presence of a graphite shell on Cu particles in the Cu–C film. Furthermore, the Cu–C/PI composite film shows better thermal properties than Cu/PI. This work provides a one‐step method for rapidly fabricating Cu/PI and Cu–C/PI composites with good thermal stability and conductivity.
Effects of cultivars and nitrogen management on wheat grain yield and protein
Authors: Ghimire Deepak , Das Saurav , Mueller Nathan D. , Creech Cody F. , Santra Dipak , Baenziger P. Stephen , Easterly Amanda C. , Maust Brian , Maharjan Bijesh
Published: July 2021

Low grain protein in hard red winter (HRW) wheat (Triticum aestivum L.) is a serious challenge for rainfed wheat growers, particularly in years with elevated grain yield. Proper nitrogen (N) management with adequate N rate and application timing is critical for optimizing grain yield and protein content. This 2‐yr experiment evaluated the effects of different N rates and application timings (fall, spring, and split) on grain yield and protein of two HRW wheat cultivars. Field studies were conducted at four different sites across Nebraska under rainfed conditions in 2018/2019 (Year 1) and 2019/2020 (Year 2). A split‐plot randomized complete block design with wheat cultivars as the whole plots and factorial combinations of six N rates and three application timings as the subplots was used in four replications. Grain yield was associated positively and grain protein negatively with the water supply to demand ratio (WS/WD) in the season. Freeman cultivar yielded better in a year with higher WS/WD and a newly developed cultivar, Ruth, yielded better in a lower WS/WD year. Nitrogen fertilization significantly increased grain yield in the site‐years with moderately higher WS/WD. There was an increase in grain protein with increasing N rates at all site‐years. Spring and split‐applied N resulted in better grain yield than fall application in the site‐year when there was a risk of N loss. This experiment suggested that an effective N management strategy for winter wheat should account for and be adaptable to weather variability to optimize grain yield and protein content.
Does work–family conflict play a role in the relationship between work‐related hazards and musculoskeletal pain?
Authors: Weale Victoria , Oakman Jodi , Clays Els
Published: July 2021

Background

Previous research identified an association between work–family conflict and musculoskeletal pain. This study explores how conflict arising from participating in both the work and nonwork domains might influence the relationships between musculoskeletal pain experienced by workers and particular work‐related hazards.

Methods

A cross‐sectional survey of data was collected from 401 workers in Belgium across seven organizations in the service and production sector. Subjective measures of the physical and psychosocial work environments, and musculoskeletal pain, were collected by questionnaire, using either an online or paper version. Moderation analyses were conducted using PROCESS v. 3.5 and SPSS v. 26.

Results

Work‐related hazards appeared to directly influence musculoskeletal pain differently for men and women. In addition, low levels of work–family conflict and supervisor support combined interactively to reduce low back pain in women. For men, the combination of high physical demands and relatively high work–family conflict were associated with increased levels of low back pain.

Conclusions

Addressing the work–life interface is worthy of further investigation as a legitimate means through which musculoskeletal pain can be reduced. Musculoskeletal risk management strategies need to include risk factors related to the work–life interface, along with those related to physical and psychosocial work‐related hazards.
PLXNA2 as a candidate gene in patients with intellectual disability
Authors: Altuame Fadie D. , Shamseldin Hanan E. , Albatti Turki H. , Hashem Mais , Ewida Nour , Abdulwahab Firdous , Alkuraya Fowzan S.
Published: July 2021

Intellectual disability (ID) is one of the most common disabilities in humans. In an effort to contribute to the expanding genetic landscape of ID, we describe a novel autosomal recessive ID candidate gene. Combined autozygome/exome analysis was performed in two unrelated consanguineous families with ID. Each of the two families had a novel homozygous likely deleterious variant in PLXNA2 and displayed the core phenotype of ID. PLXNA2 belongs to a family of transmembrane proteins that function as semaphorin receptors. Sema5A‐PlexinA2 is known to regulate brain development in mouse, and Plxna2−/− mice display defective associative learning, sociability, and sensorimotor gating. We note the existence of variability in the phenotype among the three patients, including the existence of variable degree of ID, ranging from borderline intellectual functioning to moderate–severe ID, and the presence of cardiac anomalies in only one of the patients. We propose incomplete penetrance as a possible explanation of the observed difference in phenotypes. Future cases will be needed to support the proposed link between PLXNA2 and ID in humans.
Asymmetric Synthesis of Enantiopure Pyrrolidines by C(sp3)−H Amination of Hydrocarbons
Authors: Lazib Yanis , Retailleau Pascal , Saget Tanguy , Darses Benjamin , Dauban Philippe
Published: July 2021

The asymmetric synthesis of enantiopure pyrrolidines is reported via a streamlined strategy relying on two sequential C−H functionalizations of simple hydrocarbons. The first step is a regio‐ and stereoselective catalytic nitrene C−H insertion. Then, a subsequent diastereoselective cyclization involving a 1,5‐hydrogen atom transfer (HAT) from a N‐centered radical leads to the formation of pyrrolidines that can then be converted to their free NH‐derivatives.
High‐Pressure CO Electroreduction at Silver Produces Ethanol and Propanol
Authors: Raaijman Stefan J. , Schellekens Maarten P. , Corbett Paul J. , Koper Marc T. M.
Published: July 2021

Reducing CO2to long‐chain carbon products is attractive considering such products are typically more valuable than shorter ones. However, the best electrocatalyst for making such products from CO2, copper, lacks selectivity. By studying alternate C2+producing catalysts we can increase our mechanistic understanding, which is beneficial for improving catalyst performance. Therefore, we investigate CO reduction on silver, as density functional theory (DFT) results predict it to be good at forming ethanol. To address the current disagreement between DFT and experimental results (ethanol vs. no ethanol), we investigated CO reduction at higher surface coverage (by increasing pressure) to ascertain if desorption effects can explain the discrepancy. In terms of product trends, our results agree with the DFT‐proposed acetaldehyde‐like intermediate, yielding ethanol and propanol as C2+products—making the CO2electrochemistry of silver very similar to that of copper at sufficiently high coverage.
Preparation and characterization of non‐vulcanized natural rubber‐based cocoa pod husk composites
Authors: Edjenguele Adolphe , Alegria Angel , Arrese‐Igor Silvia , Ehabe Eugene E. , Nkengafac Njukeng J.
Published: July 2021

Several ingredients are often introduced into rubber matrices during the production of natural rubber (NR) based articles, essentially to modify and improve upon the properties of the final products as well as to reduce their costs. In this study, composites of natural rubber and cocoa pod husk were prepared by solution casting and tested for their rheological and thermal properties in order to ascertain the suitability of the latter as a substitute for standard fillers in the NR industry. An increase in the level of substitution significantly reduced the rheological properties of the composites in the linear viscoelastic region but had no significant effects on their thermal stability, glass transition temperature and on the α‐relaxation of the NR composites except at the highest level of substitution (25%). However, α‐relaxation and rheological shift factors were always temperature‐dependent and had a direct influence on their electrical conductivity with no permanent chemical bonds formed or broken during preparation of the composites. Hence, untreated cocoa pod husk could serve as non‐reinforcing filler for raw natural rubber.