bluuebunny/crossref_metadata_2025 huggingface dataset:
Smartphone screen testing, a novel pre-diagnostic method to identify SARS-CoV-2 infectious individuals
Authors: Young Rodrigo M , Solis Camila J , Barriga-Fehrman Andres , Abogabir Carlos , Thadani Alvaro R , Labarca Mariana , Bustamante Eva , Tapia Cecilia V , Sarda Antonia G , Sepulveda Francisca , Pozas Nadia , Cerpa Leslie C , Lavanderos María A , Varela Nelson M , Santibañez Alvaro , Sandino Ana M , Reyes-Lopez Felipe , Dixon Garth , Quiñones Luis A
Published: June 2021

The COVID-19 pandemic will likely take years to control globally, and constant epidemic surveillance will be required to limit the spread of SARS-CoV-2, especially considering the emergence of new variants that could hamper the effect of vaccination efforts. We developed a simple and robust – Phone Screen Testing (PoST) – method to detect SARS-CoV-2-positive individuals by RT-PCR testing of smartphone screen swab samples. We show that 81.3–100% of individuals with high-viral-load SARS-CoV-2 nasopharyngeal-positive samples also test positive for PoST, suggesting this method is effective in identifying COVID-19 contagious individuals. Furthermore, we successfully identified polymorphisms associated with SARS-CoV-2 Alpha, Beta, and Gamma variants, in SARS-CoV-2-positive PoST samples. Overall, we report that PoST is a new non-invasive, cost-effective, and easy-to-implement smartphone-based smart alternative for SARS-CoV-2 testing, which could help to contain COVID-19 outbreaks and identification of variants of concern in the years to come.
”Det blir ofte vanskelig å få til de tverrfaglige greiene”
Authors: Dagsland Torunn Paulsen
Published: June 2021

Den nye forskriften overordnet del – verdier og prinsipper for grunnopplæringen knytter begrepet tverrfaglighet til tre ulike temaer og fremhever det som et pedagogisk prinsipp for undervisningen i Norge. Artikkelen belyser hvordan denne delen av læreplanverket beskriver begrepet tverrfaglig som pedagogisk prinsipp. Undersøkelsen som ligger til grunn for artikkelen, viser at læreplanen åpner for at lærere kan konstruere sin egen forståelse av hva tverrfaglig tilnærming er, og dermed kan praktisere ulike former for tverrfaglig opplæring. Artikkelen beskriver hva et utvalg av faglærere på ungdomstrinnet (i naturfag og kunst og håndverk) legger i begrepet tverrfaglig slik de forstår bruken i læreplanverket. Videre tar den for seg hva disse lærerne oppfatter som en tverrfaglig tilnærming i sin egen operasjonaliserte undervisning. Flertallet av disse lærerne hevder at tverrfaglighet sett i lys av overordnet del innebærer en undervisning hvor fagene integreres og glir over i hverandre. Imidlertid viser undersøkelsen at når de fleste av informantene ser på sin egen operasjonaliserte undervisning, både forstår de og utøver de den tverrfaglige tilnærmingen annerledes. Eksempler lærerne trekker fram fra egen undervisning, illustrerer at den tverrfaglige tilnærmingen til undervisning både forstås og praktiseres på ulike måter med ulike grader av tverrfaglighet. Artikkelen ser også på i hvilken grad lærerne har erfaring med undervisning på tvers av naturfag og faget kunst og håndverk. Lærerne i denne undersøkelsen vedgår at de ikke har erfaring med tverrfaglig samarbeid mellom disse fagene. Undersøkelsen bygger på en dokumentanalyse og intervjuer av tolv lærere i ungdomsskolen som underviser i naturfag og faget kunst og håndverk. Teoretisk rammeverk for undersøkelsen har vært Goodlad (1977, 1979) sin konseptuelle teori om læreplanens ulike fremtredelsesformer.
Nøkkelord: tverrfaglig, tverrfaglig tilnærming, den formelle plan, den oppfattede plan, den operasjonaliserte plan, den erfarte plan og samarbeid på tvers av naturfag og kunst og håndverk.
Microbiological Profile and Sensitivity Data among the Neonatal Population from a Tertiary Care Hospital in South India
Authors: Reshad Mohamed , Binesh Balachandran , Sharief Moideen , Shad Muheidheen
Published: June 2021

Background: Sepsis is one of the most common causes of mortality and morbidity among neonates. The prevalence of microorganisms varies with regions, and the sensitivity pattern also changes over time.  

Aim: To study the prevalence of various microorganisms and their sensitivity pattern among the neonatal population in a tertiary care hospital in South India.

Methods: This was a retrospective descriptive study. We screened the records of all neonates admitted to the hospital during a period starting from January 1st, 2016, to September 30th, 2018. The details of the patients with any sterile body fluids showing microbial growth were collected. Descriptive analysis was done using IBM SPSS version 20.

Results: There were 77 organisms isolated during the study period, with the majority among outborn babies. Seventy-two isolates were from blood, two from CSF and urine, with one from the central line. The most common organism was Klebsiella (22%), followed by Burkholderia cepacia (12%), Staphylococcus aureus (9%), and Streptococcus agalactiae (7%). 9% of the organisms were fungus, with the most common being Kodamaea Ohmeri. Among the antibiotics tested for sensitivity, Meropenem had the maximum sensitivity ratio (84%), followed by Co-trimoxazole (83%) and Vancomycin (82%). Piperacillin tazobactam sensitivity was only 56%, and Ciprofloxacin was sensitive in 65% of cases. Most (6 out of 7) of the fungal isolates were sensitive to fluconazole.

Conclusions: Klebsiella remains to be the most common organism causing severe infection in young infants. Worrying rise in the incidence of uncommon organisms (like Kodamaea Ohmeri, GBS, Burkholderia) and carbapenem resistance was noted.  It is, therefore, essential to update the antibiotic policy of the neonatal units on a timely basis.
Integrated Pest Management In Coffee
Authors: Oliveira Jeniffer Ribeiro De , Santana Weslley do Rosário , Altoé Jalille Amim , Carrion Paula Abiko Navarro , Baldan Winy Galacho , Lima Alex Silva , Rosa Luã Víthor Chixaro Almeida Falcão , Gomes Raquel da Silva , Cavalcanti André Cayô , Nascimento Alan de Lima , Oliveira Vinícius de Souza
Published: June 2021

Coffee (Coffea sp.) Is a crop of great economic relevance, it stands out as one of the most important agricultural commodities for the country. As with any other high value-added crop, coffee crops suffer from the attack of many pests, and proper management of these pests is critical to successful production. Integrated Pest Management (IPM) is a pest management system that associates the environment and population dynamics of the pest, considers the use of all available plant protection methods and the integration of appropriate measures to maintain the population level of thepest below the level of damage in an economically, environmentally and ecologically viable way. The purpose of this literature review was to gather information on research involving the integrated management of pests in the coffee crop.
Effects of Bank Charges on Bank's Customers Saving and Income in Nigeria
Authors: Dairo Ridwan , Joseph Tonuchi E.
Published: June 2021

Effective saving mobilization by the deposit money banks is a perquisite to achieve the macroeconomic objectives of price stability, full sustainable economic growth, full employment, and exchange rate stability. This study therefore investigates effect of bank charges on saving and income of bank’s customers in Nigeria. The study sampled 180 bank customers in Lagos state, and it was revealed that excessive bank charges is not only hindering effective saving mobilization in the country but also the financial inclusion policy of the Central Bank of Nigeria. It was further discovered that excessive bank charges are of greater concern to small savers like students and self-employed customers. The study therefore concludes that excessive and indiscriminate bank charges does not only cause more than 30 percent of Nigerian bank customers to switch to other banks but impacts their saving culture and income.
Aflatoxin B1: Cytotoxic mode of action evaluated by mammalian cell cultures
Authors: Scaife J.F.
Published: July 2002

Aflaxton B1 rapidly inhibits RNA synthesis in rat liver cells, slices or liver in vivo. Established human cells lines (kidney T‐cells, HeLa S3, Chang liver) and mouse fibroblast 3t3 are more slowly affected. Prolonged exposure of synchronized cell cultures to the agent show that cells are retarded in their passage through the S‐phase and exhibit a decreased rate of DNA synthesis. Consequent to this, mitosis is also inhibited. Liver cells appear to convert aflatoxin B1 to a more potent cytotoxin which can then affect normally non‐susceptible cells. This may explain the susceptibility of liver to tumorogenesis by this carcinogen.
Renal transport and disposition of Na‐2‐mercaptoethane sulfonate disulfide (Dimesna) in the rat
Authors: Ormstad K. , Uehara N.
Published: July 2002

The transport and reduction of dimesna (NA‐2‐mercaptoethane sulfonate disulfide) was studied in vitro using isolated, perfused rat kidney, and isolated renal epithelial cells. Cellular uptake of dimesna was found to be dependent on an active transport mechanism working across the luminal brush border, with an app. K
m of ∼22 μM and V
max ∼1.4 nmol. 106 cells−1.min−1. Among other low molecular thiols or disulfides reduced glutathione was the only one to exert competitive inhibition. γ‐GT‐activity or cellular GSH status had no influence on renal uptake of dimesna, but the intracellular reduction rate was dependent on access to reduced glutathione as a cofactor.
Formation of lipid—protein bilayers by micropipette guided contact of two monolayers
Authors: Schuerholz Theo , Schindler Hansgeorg
Published: July 2002

Bilayers of a few μm2 area were formed from lipid‐protein vesicles. First, two monolayers are generated from vesicles, which then are brought into local contact by use of a micro‐pipette to form a bilayer. Membrane channels of Matrix—Protein and of Colicin A exhibit normal properties in these bilayers. Apart from avoiding solvents completely and of using characterized vesicles as starting materials there are two main virtues to this strategy: (1) Current resolution is very high, a few tenths of a pA at 150 μs time resolution; and (2) transport processes can be studied in dependence of surface pressure between 28 and 48 mN/m.
Use of the fluorescent pH probe pyranine to detect heterogeneous directions of proton movement in bacteriorhodopsin reconstituted large liposomes
Authors: Seigneuret Michel , Rigaud Jean-Louis
Published: July 2002

Light‐induced proton pumping was measured in bacteriorhodopsin reconstituted large proteoliposomes (prepared by reverse‐phase evaporation) using the fluorescence of internally trapped pyranine. From the effect of the one‐sided inhibitor La3+, the presence of both inside‐out and right‐side‐out oriented bacterio‐rhodopsin was demonstrated. The use of pyranine at acidic pH allowed us to detect two distinct populations of proteoliposomes which exhibited oppositely directed proton pumping. This suggests that the two orientations, of bacteriorhodopsin are at least partially distributed in different proteoliposomes.
The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits
Authors: Hucho Ferdinand , Oberthür Walter , Lottspeich Friedrich
Published: July 2002

A binding site for the channel‐blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the α‐, β‐ and δ‐chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, α 248, β 254, and δ 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position δ 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.
A cDNA clone encoding the precursor for a 10.2 kDa photosystem I polypeptide of barley
Authors: Okkels Jens Sigurd , Scheller Henrik Vibe , Jepsen Lars Bæk , Møller Birger Lindberg
Published: July 2002

Two cDNA clones for the barley photosystem I polypeptide which migrates with an apparent molecular mass of 9.5 kDa on SDS‐polyacrylamide gels have been isolated using antibodies and an oligonucleotide probe. The determined N‐terminal amino acid sequence for the mature polypeptide confirms the identification of the clones. The 644 base‐pair sequence of one of the clones contains one large open reading frame coding for a 14 882 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 193 Da. The hydropathy plot of the polypeptide shows one membrane‐spanning region with a predicted α‐helix secondary structure. The gene for the 9.5 kDa polypeptide has been designated PsaH.
μ type opioid receptors in rat periaqueductal gray‐enriched P2 membrane are coupled to G‐protein‐mediated inhibition of adenylyl cyclase
Authors: Fedynyshyn J.P. , Lee N.M.
Published: July 2002

The periaqueductal gray (PAG) region of the midbrain has been implicated in both stimulation‐produced and opioid‐induced analgesia. High‐affinity μu‐selective opioid‐binding sites associated with μu type opioid receptors have been detected in rat PAG‐enriched P2, membranes, and these receptors have been shown to be coupled to guanine nucleotidebinding proteins (G‐proteins). In the present study the potential G‐protein‐mediated coupling of μu type opioid receptors to the inhibition of adenylyl cyclase was examined utilizing in vitro adenylyl cyclase assays. In the presence of Na+, opioid agonists inhibited adenylyl cyclase in aμu selective, naloxone reversible, dose dependent, and pertussis toxin sensitive manner. Overall the data suggests that μu type opioid receptors in the rat PAG are coupled to G‐protein‐mediated inhibition of adenylyl cyclase.
Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4‐2J
Authors: Buscail Louis , Gourlet Philippe , Cauvin Annick , De Neef Philippe , Gossen Denis , Arimura Akira , Miyata Atsuro , Coy David H. , Robberecht Patrick , Christophe Jean
Published: July 2002

We characterized highly selective receptors for PACAP, the pituitary adenylate cyclase activating peptide, in the tumoral acinar cell line AR 4‐2J derived from the rat pancreas. PACAP, a novel hypothalamic peptide related to vasoactive intestinal peptide (VIP), was tested as the full natural 38‐residue peptide (PACAP‐38) and as an N‐terminal amidated 27‐residue derivative (PACAP‐27). The binding sites showed considerable affinity for [125I]PACAP‐27 (K
d =0.4 nM) and PACAP‐38, while their affiity for VIP and the parent peptide helodemin was 1000‐fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP‐38 and PACAP‐27 (K
act = 0.2 nM) being much higher than that of VIP (K
act= 100 nM) and helodemin (K
act = 30 nM). Chemical cross‐linking of [125I]PACAP‐27 followed by SDS‐PAGE and autoradiography revealed a specifically cross‐linked peptide with an M
r, of 68000 (including 3000 for one PACAP‐27 molecule).
Refilling of endothelial calcium stores without bypassing the cytosol
Authors: Lückhoff Andreas , Busse Rudi
Published: July 2002

The present study was undertaken to define the route of Ca2+ used for refilling of intracellular Ca2+ stores in endothelial cells. Ca2+ stores, after emptying with bradykinin in Ca2+ free solution and termination of the stimulation with the bradykinin antagonist, Hoe 140, were allowed to refill by addition of Ca2+. Refilling was prevented by 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone (BuBHQ), an inhibitor of microsomal Ca2+ sequestration. BuBHQ induced large increases in the cytosolic Ca2+ concentration during the refilling phase. This finding is not compatible with a model proposing Ca2+ uptake into the stores directly from the extracellular space but provides evidence for uptake from the cytosolic compartment.
15N nuclear magnetic resonance studies of the B domain of Staphylococcal protein A: sequence specific assignments of the imide 15N resonances of the proline residues and the interaction with human immunoglobulin G
Authors: Torigoe Hidetaka , Shimada Ichio , Waelchli Markus , Saito Akiko , Sato Moriyuki , Arata Yoji
Published: July 2002

15N nuclear magnetic resonance (NMR) studies of the B domain (FB) of Staphylococcus protein A, which is uniformly labeled with 15N, are reported. The α CH(i)‐15N(i) connectivity in the 1H‐15N HMBC spectrum and the 13C(i‐1)‐15N(i) spin coupling in the 15N spectrum of a 13C‐, 15N‐doubly labeled FB were used to establish the assignments of the imide 15N resonances for all the three Pro residues that exist in FB. Addition of human IgG caused a significant downfield shift of the Pro‐39 resonance. This result is quite consistent with our previous suggestion that a significant conformation change is induced in the Ser‐42‐Ala‐55 helical region of FB when it is bound to human IgG.
Effect of formate on Mössbauer parameters of the non‐heme iron of PS II particles of cyanobacteria
Authors: Semin B.K. , Loviagina E.R. , Aleksandrov A.Yu. , Kaurov Yu.N. , Novakova A.A.
Published: July 2002

Mössbauer spectra were measured for PSII particles having an active water‐splitting system. The particles were isolated from the thennophilic cyanobacterium Synechococcus elongatus enriched in57Fe. The Mössbauer resonance absorption spectrum is a superposition of 3 doublets with the following quadrupole splitting and chemical shift: 1, δ = 0.40, Δ = 0.85; II, δ = 1.35,Δ =2.35; III, δ = 0.25, Δ = 1.65. The δ and Δ values of doublets I, II, III are characteristic of proteins with iron‐sulphur center, non‐heme iron of the reaction center of higher plants and of the oxidized cytochrome 6–559. Treatment with sodium formate to remove bicarbonate affects only the doublet of non‐heme iron, causing its quadrupole splitting to reduce to 1.75 and the chemical shift to reduce to 0.90. After washing out the formate, the Mossbauer spectrum of non‐heme iron is restored. The data suggest that bicarbonate is a ligand for the non‐heme iron of the reaction center of cyanobacteria.
Bacterial expression of human muscarinic receptor fusion proteins and generation of subtype‐specific antisera
Authors: Levey Allan I. , Stormann Tom M. , Brann Mark R.
Published: July 2002

A family of five muscarinic acetylcholine receptor genes (m1–m5) encode highly related proteins; however, for methodological reasons it has not been possible to detect the gene product individually. To develop antibody probes specific for the receptor subtypes, unique regions of m1–m5 cDNAs, corresponding to the third cytoplasmic (13) loops, were subcloned into bacterial expression vectors and the fusion proteins expressed in E. coli were used to generate rabbit antisera. These antisera react specifically with the respective fusion proteins on immunoblots and selectively immunoprecipitate each of the native cloned receptors. Since the 13 loops are immunogenic and the epitopes in the cloned receptors are accessible to antibodies, this approach should be valuable for immunological studies of the native receptors.
Modulation of protease specificity by a change in the enzyme microenvironment Selectivity modification on a model substrate, purified soluble proteins and gluten
Authors: Hertmanni Patricia , Picque Eric , Thomas Daniel , Larreta-Garde Véronique
Published: July 2002

Subtilisin BPN activity on a synthetic substrate is found to decrease with the concentration of soluble additives such as sugars and polyols, the catalytic efficiency of the enzyme being related to the water activity in the reaction medium. Limited hydrolysis of B chain of insulin is followed and the cleavage priority determined. When carried out in glycerol‐containing medium, both enzyme catalytic behavior and specificity are perturbed: a different cleavage order and a selectivity restriction are observed. The experiments were generalised to purified proteins and to an insoluble protein complex. The hydrolysis kinetics of purified gliadins by pepsin and of gluten by a Bacillus neutral protease are modulated in presence of water activity depressors. Glycerol is able to increase both pepsin efficiency and gluten protein solubility. The hydrolysis order is affected by water‐structuring molecules in the enzyme microenvironment and new peptides appear whatever the size and initial solubility of the substrate.
Amylin injection causes elevated plasma lactate and glucose in the rat
Authors: Young Andrew A. , Wang Ming-Wei , Cooper Garth J.S.
Published: July 2002

Intravenous injections of 25.5 nmol rat amylin into fasted anesthetized rats caused a rapid increase in plasma lactate followed by an increase in plasma glucose; there was a transient fall in blood pressure. Subcutaneous injection of 25.5 nmol amylin also caused increases in lactate and glucose but did not change blood pressure. Similar responses were observed during somatostatin infusion and in the absence of changes in catecholamines. These results fit with a scheme in which amylin elicits muscle glycogenolysis, release of lactate, and increased hepatic gluconeogenesis due to increased supply of substrate.
Okadaic acid stimulates carnitine palmitoyltransferase I activity and palmitate oxidation in isolated rat hepatocytes
Authors: Guzmán Manuel , Castro José
Published: July 2002

Okadaic acid increased in parallel with carnitine palmitoyltransferase I activity and the rate of palmitate oxidation in isolated rat hepatocytes. Nevertheless, okadaic acid had no significant effect on the rate of octanoate oxidation. Maximal effects of okadaic acid were similar and non‐additive to those of dibutyryl‐cAMP, forskolin and glucagon. Results indicate that carnitine palmitoyltransferase I activity may be controlled by a mechanism of phosphorylation/dephosphorylation.
Mechanism of maturation and nature of carbohydrate chains of boar sperm acrosin
Authors: Moos J. , Tesarik J. , Leca G. , Peknicova J.
Published: July 2002

The acrosin zymogen proacrosin exists in two molecular forms which are believed to be single‐chain polypeptides. During autoactivation in a cell‐free system, the 55 and 53 kDa zymogens are sequentially converted into the 49, 36, 31 and 25 kDa forms. A similar mechanism of maturation was revealed, when the calcium ionophore A23187 was added to suspensions of boar spermatozoa. The 49 kDa form has been identified as the first active acrosin form in the maturation cascade. However, this form is indistinguishable from the 53 kDa zymogen in SDS‐PAGE at nonreducing conditions. Two carbohydrate chains were evidenced on the acrosin molecule. The chain attached to the Asn3 of the acrosin light chain was enzymatically cleaved without loss of acrosin activity. By contrast, the carbohydrate chain linked to the acrosin heavy chain could be cleaved only after acrosin denaturation. Based on the susceptibility of acrosin to endoglycosidases F and H, a biantennary structure of both carbohydrate chains is proposed.
Analysis of filamin and α‐actinin binding to actin by the stopped flow method
Authors: Goldmann W.H. , Isenberg G.
Published: July 2002

We ascertained by the stopped flow method the overall association rate constant, k
+1, of filamin and α‐actinin to fluorescently labelled filamentous actin of ~ 1.3 × 106M−1 · s−1 and ~ 1.0 × 106M−1 · s−1 as well as the overall dissociation rate constant,k
−1 of ~ 0.6s−1 and ~ 0.4s−1, respectively. The overall equilibrium constant, K, for filamin and α‐actinin to actin deduced from the relation K = agree well with published data.
Uridylylation of the PII protein in Rhizobium leguminosarum
Authors: Colonna-Romano Sergio , Patriarca Eduardo Jorge , Amar Mohamed , Bernard Paola , Manco Giuseppe , Lamberti Alessandro , laccarino Maurizio , Defez Roberto
Published: July 2002

Permeabilization with cetyl trimethyl ammonium bromide was used to study the post‐translational modification of the PII protein in Rhizobium leguminosarum. Upon incubation with radioactive UTP a single band was obtained after SDS‐PAGE and autoradiography. RNase resistance and snake venom phosphodiesterase sensivity showed that radioactivity was bound through a phosphodiester bond to a protein which was absorbed by an antiserum specific for the PII protein. Uridylylation of the PII protein was shown to be dependent on the modifications of the glutamine/α‐ketoglutarate ratio.
Cyclic disulfide model of the major antigenic site of serotype‐C foot‐and‐mouth disease virus
Authors: Camarero Julio A. , Andreu David , Cairó Jordi J. , Mateu Mauricio G. , Domingo Esteban , Giralt Ernest
Published: July 2002

A cyclic disulfide peptide representing antigenic site A of foot‐and‐mouth‐disease virus (FMDV) strain C‐S8cl (residues 134 to 155 of viral protein l (VP1) with Tyr136 and Arg153 replaced by cystine; TTCTASARGDLAHLTTTHACHL) was synthesized by solid phase methods. Formation of the cyclic disulfide was carried out by air oxidation of the fully deprotected and reduced bis‐cysteine precursor, under high dilution conditions. The identity of the cyclic peptide was confirmed by both physical and enzymatic methods. A conformational study of the cyclic peptide and of its linear parent structure (YTASARGDLAHLTTTHARHLP, residues 136–156 of VP1 of FMDV C‐S8cl) by circular dichroism in the presence of a structure‐inducing solvent showed the cyclic disulfide analog to adopt lower levels of α‐helix than its linear counterpart. In competitive ELISA assays both peptides reacted with similar affinity against a representative panel of neutralizing monoclonal antibodies directed towards antigenic site A. Thus, a high inherent flexibility of this loop may preclude a conformational restriction strong enough to alter recognition by anti‐virus antibodies.
Coupling of D2 and D3 dopamine receptors to G‐proteins
Authors: Castro Sandra W. , Strange Philip G.
Published: July 2002

Recombinant cell lines expressing D2 and D3 dopamine receptor isoforms have been used to study coupling of these receptors to G‐proteins from the effects of GTP to reduce agonist binding affinities in agonist/[3H]spiperone competition experiments. D2(short) expressed in Ltk− cells, D2(long) expressed in Ltk− or CHO cells and D3 expressed in CHO cells all showed coupling to the endogenous G‐proteins of the cells. The detailed agonist binding characteristics indicated that there were differences in receptor/G‐protein coupling for the same receptor (D2(long) expressed in two cell types or for different receptors (D2(long),D2(short) expressed in the same cell type.
Conserved secondary structures in the ITS2 of trematode pre‐rRNA
Authors: Michot Bernard , Despres Laurence , Bonhomme François , Bachellerie Jean-Pierre
Published: July 2002

Biological functions of transcribed spacer regions in eucaryotic pre‐rRNAs remain elusive so far. Utilization of the comparative approach to analyse their secondary structure has been impeded by the extensive sequence divergence observed among most of the specimens available to date. However, we have taken advantage of the recent derivation of a set of largely similar sequences for different schistosome species to look for the presence of constrained secondary structures within the internally transcribed spacer 2 (ITS2). We show that several common features of secondary structure are shared by these species despite sequence variation, with some of them supported by compensatory changes, suggesting a significant role of ITS2 as an RNA domain during ribosome biogenesis.
The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin‐assisted folding
Authors: Staniforth Rosemary A. , Cortés Antonio , Burston Steven G. , Atkinson Tony , Holbrook J.John , Clarke Anthony R.
Published: July 2002

mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding. The hydrophilic and stable cMDH is bound by cpn60 but released by MG‐ATP alone, while the hydrophobic and unstable mMDH requires both Mg‐ATP and cpn 10. Citrate equalises the stability of the native state of the two proteins but has no effect on the co‐chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor. The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg‐ATP. In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin‐assisted folding is 3–4‐times faster.
Homology modelling and 1H NMR studies of human leukaemia inhibitory factor
Authors: Smith David K. , Treutlein Herbert R. , Maurer Till , Owczarek Catherine M. , Layton Meredith J. , Nicola Nicos A. , Norton Raymond S.
Published: July 2002

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony‐stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with 1H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pK
a values of which range from 3.6 to 7.4. These pK
a values were assigned to individual histidine residues from NMR studies on a series of His → Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.
Specific binding sites for 3,3′‐diiodo‐l‐thyronine (3,3′‐T2) in rat liver mitochondria
Authors: Lanni Antonia , Moreno Maria , Horst Claus , Lombardi Assunta , Fernando Goglia
Published: July 2002

Specific binding sites for 3,3′‐T2 can be detected in swollen and osmotically treated mitochondria (OTM) from normal and hypothyroid rat liver. In hypothyroid animals, maximal values of binding were obtained at 0°C and 20°C while values were lower at 37°C and no specific binding could be observed at 60°C. Binding was maximal at pH 6.4 and the mean values for the apparent association constant (K
a) and the binding capacity were on average 0.5 × 108 M−1 and 2.4 pmol/mg mitochondrial protein, respectively. No differences were observed between normal and hypothyroid rats with the exception of the capacity that was higher in normal animals (5.5 pmol/mg mitochondrial protein). The specificity of 3,3′‐T2 binding, examined in competition studies, followed this order: 3,3′‐T2 > 3,5‐T2 > rT3. The other iodothyronines (3′,5′‐T2, T3, T4, 3‐T1, 3′‐T1, 3,5‐Diac and 3,3′‐Diac) showed only a small competition or none at all.
The H19 gene is expressed within both epithelial and stromal components of human invasive adenocarcinomas
Authors: Dugimont Thierry , Curgy Jean‐Jacques , Wernert Nicolas , Delobelle Anne , Raes Marie‐Berthe , Joubel Anita , Stehelin Dominique , Coll Jean
Published: July 2002

Summary— In a previous work, we have isolated the human H19 gene and shown accumulation of transcripts in various human tumors including breast carcinomas (Douc‐Rasy et al (1993) Int J Oncol 2, 753–758). Questions arose, after Northern blot results, about the precise H19 mRNA location, specially in normal breast tissues and benign or malign primary breast tumors. Then we performed molecular in situ hybridization to get insight into tissue expression of the H19 gene. Examined resections included one normal tissue, one fibroadenoma and 13 cancers. Results obtained with the H19 probe can be summarized as follows: 1) in normal breast tissues signals were focally observed in epithelial cells, but more predominantly in the palleal tissue which is sensitive to hormones; 2) in the fibroadenoma, fibroblastic cells were extensively labeled at the stroma‐epithelium boundary, but epithelial cells were negative; and 3) in primary cancers, eight specimens exhibited signals on stromal cells, one specimen on epithelial cells and four on both epithelial and stromal cells. Data provide the following evidence: 1) usually labeled cells are clustered, either within normal or pathological tissues; 2) the labeling pattern highly differs from one tumor to another; and 3) H19 probe displays very different signals from one cell to another in a given compartment of a given tissue section. In conclusion, it seems that a high H19 expression matches the tumor invasion. Our results suggest that the expression of this gene is concerned by the relationships between epithelial and stromal cells, and can reflect peculiar physiological states of the cells. Furthermore, we discuss results showing an abundant expression of H19 gene in some adenocarcinomas of bad prognosis, in the context of the otherwise established tumor‐suppressor role of this gene, or the strictly controlled gene dosage, which could be overridden in these particular cases.
The ability to re‐express polysialylated NCAM in soleus muscle after denervation is reduced in aged rats compared to young adult rats
Authors: Olsen Marianne , Zuber Christian , Roth Jürgen , Linnemann Dorte , Bock Elisabeth
Published: July 2002

The neural cell‐adhesion molecule, NCAM, contains an unusual homopolymer of sialic acid units, polysialic acid. This carbohydrate seems to be involved in neurite outgrowth, bundling and branching, processes which are important during reinnervation. In aged rats, reinnervation of denervated muscle fibres is incomplete. In this study, age‐related changes in the degree of polysialylation of NCAM re‐expressed after denervation were examined using a monoclonal antibody recognizing polysialic acid and a polyclonal antibody recognizing NCAM. The results show that, after denervation, the degree of polysialylation on NCAM was clearly reduced in rat soleus muscle of aged, compared to young, adult rats. This age‐related change in expression of polysialic acid probably influences the reinnervation process in aged muscle.
Ontogenic development of membrane lipids in the chick optic lobe
Authors: Rivas Emilio A. , Fernández‐Tomé Maria Del Carmen , Biancotti Juan C. , Sterin‐Spezia Norma B. , de Plazas Sara Fiszer
Published: July 2002

The developmental profiles of the lipid composition and their de novo synthesis and remodelling in the optic lobe of the chicken were studied. The 32P incorporation to phospholipids showed an active de novo synthesis mainly of phosphatidylinositol and of a particular fraction of phosphatidylcholine during the early stages of the embryo development, concomitantly with the beginning of synaptogenesis. This de novo synthesis of phospholipids strongly increased at hatching. On the other hand, phosphatidylinositol presented an active lipid exchange (acylation‐leacylation) in the early stages of embryogenesis, indicating a strong incorporation of 14C‐arachidonic acid during this period, followed by a fast drop in specific activity. Two different fractions of phosphatidylcholine were isolated by high‐performance thin‐layer chromatography with a different profile of fatty acid composition, disclosing their different physicochemical behavior, metabolic activities and evolution during embryogenesis. 32P incorporation into phosphatidylethanolamine remained very low during the earliest stages of embryogenesis, showing an increase when the process of synaptogenesis began, until hatching, when radioactivity reached a plateau. 14C‐arachidonic acid incorporation into phosphatidylethanolamine was minimal. Furthermore, the phosphatidylethanolamine pool was progressively enriched in its ethanolamine plasmalogen throughout the development. Chromatographic analysis of lipid extracts showed the presence of cerebroside traces after 16 days of embryo incubation. At hatching, a remarkable increase in non‐hydroxylated cerebrosides was observed concurrently with the appearance of hydroxylated ones. These glycosphingolipids, as well as the sulfatides, were markedly increased in the lipid extracts of optic lobes of adult animals, indicating the progressive development and maturity of the myelin sheath.
Interaction of wild‐type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles
Authors: Preikschat Petra , Kazaks Andris , Dishlers Andris , Pumpens Paul , Krüger Detlev H. , Meisel Helga
Published: July 2002

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild‐type (wt) and in‐frame deleted variants of the HBV core gene has been identified as a typical feature of HBV‐infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two‐vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co‐expression of wt and a mutant core lacking 17 amino acid residues (77–93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self‐assembly.
Zinc ions inhibit oxidation of cytochrome c oxidase by oxygen
Authors: Aagaard Anna , Brzezinski Peter
Published: July 2002

Cytochrome c oxidase is a membrane‐bound enzyme that catalyses the reduction of O2 to H2O and uses part of the energy released in this reaction to pump protons across the membrane. We have investigated the effect of addition of Zn2+ on the kinetics of two reaction steps in cytochrome c oxidase that are associated with proton pumping; the peroxy to oxo‐ferryl (Pr→F) and the oxo‐ferryl to oxidised (F→O) transitions. The Zn2+ binding resulted in a decrease of the F→O rate from 820 s−1 (no Zn2+) to a saturating value of ∼360 s−1 with an apparent K
D of ∼2.6 μM. The Pr→F rate (∼104 s−1 before addition of Zn2+) decreased more slowly with increasing Zn2+ concentration and a K
D of ∼120 μM was observed. The effects on both kinetic phases were fully reversible upon addition of EDTA. Since both the Pr→F and F→O transitions are associated with proton uptake through the D‐pathway, a Zn2+‐binding site is likely to be located at the entry point of this pathway, where several carboxylates and histidine residues are found that may co‐ordinate Zn2+.
The C‐terminal part of VIP is important for receptor binding and activation, as evidenced by chimeric constructs of VIP/secretin
Authors: Wulff Birgitte , Møller Knudsen Sanne , Adelhorst Kim , Fahrenkrug Jan
Published: July 2002

The structural requirements of vasoactive intestinal polypeptide (VIP) for receptor binding and cAMP production were studied in a cell line stable transfected with the cDNA for rat VIP receptor 1 (rVIPR 1). Using a number of chimeric constructs of VIP and the homologue peptide secretin, it was found that the N‐terminal half of VIP (1‐11) can be exchanged with the corresponding sequences in secretin with only modest influence on binding and activation, whereas the opposite chimeras with N‐terminal VIP and C‐terminal secretin were unable to bind to the VIP receptor. The data suggest that the C‐terminal region of VIP is important for receptor binding and activation.
NADH:Fe(III)‐chelate reductase of maize roots is an active cytochrome b5 reductase
Authors: Sparla Francesca , Bagnaresi Paolo , Scagliarini Sandra , Trost Paolo
Published: July 2002

Microsomal NADH:Fe(III)‐chelate reductase (NFR) of maize roots has been purified as a monomeric flavoprotein of 32 kDa with non‐covalently bound FAD. In the presence of NADH, NFR efficiently reduced the physiological iron‐chelate Fe(III)‐citrate (K
cat/K
m(Fe(III)‐citrate)=6.0×106 M−1 s−1) with a sequential reaction mechanism. Purified NFR was totally inhibited by the sulfhydryl reagent PHMB at 10−9 M, and it could use cyt b
5 as alternative electron acceptor with a maximal reduction rate as high as with Fe(III)‐citrate. We conclude that in maize roots the reduction of Fe(III)‐citrate is chiefly performed by a cytochrome b
5 reductase, mostly associated with intracellular membranes and in part with the plasma membrane.
Generation of protein carbonyls by glycoxidation and lipoxidation reactions with autoxidation products of ascorbic acid and polyunsaturated fatty acids
Authors: Miyata Toshio , Inagi Reiko , Asahi Koichi , Yamada Yukihiro , Horie Katsunori , Sakai Hideto , Uchida Koji , Kurokawa Kiyoshi
Published: July 2002

Accumulation of carbonyl derivatives of proteins (protein carbonyl) is taken as a biomarker of oxidative protein damage in aging and in various diseases. We detected protein carbonyls in situ in human diabetic arteriosclerotic tissues and characterized the formation of protein carbonyls. Protein carbonyls were identified in the thickened intima of arterial walls and co‐localized with protein adducts formed by carbonyl amine chemistry between protein and carbonyl compounds derived from autoxidation of carbohydrates, lipids, and ascorbate, i.e. advanced glycation end products or glycoxidation products, such as carboxymethyllysine (CML) and pentosidine, and lipoxidation products, such as malondialdehyde (MDA) and 4‐hydroxy‐nonenal (HNE). In vitro incubation of proteins with ascorbic acid accelerated the production of protein carbonyls as well as CML and pentosidine, and incubation with arachidonate accelerated the production of protein carbonyls as well as CML, MDA, and HNE. By contrast, incubation of proteins with glucose resulted in the production of CML and pentosidine, but not protein carbonyls. Schiff base inhibitors, (±)‐2‐isopropylidenehydrazono‐4‐oxo‐thiazolidin‐5‐ylacetanilide and aminoguanidine, inhibited the production of protein carbonyls after incubation with ascorbate and arachidonate. The present study suggests that ascorbate and polyunsaturated fatty acids, but not glucose, represent potential sources of protein carbonyls, and that both the glycoxidation and lipoxidation reactions contribute to protein carbonyl formation in aging and various diseases.
Identification of the protein components of mismatch binding complexes in human cells using a gel‐shift assay
Authors: Nakajima Eiitsu , Ikejima Miyoko , Watanabe Atsushi , Shimada Takashi
Published: July 2002

In eukaryotes, mismatch recognition is thought to be mediated by two heterodimers, hMutSα (hMSH2+hMSH6), which preferentially binds to base‐base mismatches and hMutSβ (hMSH2+hMSH3), which binds to insertion/deletion loops. We studied these mismatch binding activities in several human cell lines with a gel‐shift assay using various mismatch oligonucleotides as substrates. Both hMutSα and hMutSβ activities could be detected in various human cell lines. In cells with amplified copies of the hMSH3 gene, a large increase in hMutSβ and a reduction in hMutSα were observed. To identify the composition of each mismatch binding complex, the protein‐DNA complexes were transferred from gel‐shift polyacrylamide gel to a polyvinylidene difluoride membrane and were subjected to immunoblot analysis with an enhanced chemiluminescence protein detection system. The results clearly demonstrated that hMutSα detected by the gel‐shift assay was composed of hMSH2 and hMSH6, while hMutSβ was composed of hMSH2 and hMSH3. Our data, therefore, support a model whereby formation of hMutSα and hMutSβ is mutually regulated. Combination of a gel‐shift assay with immunoblotting (shift‐Western assay) proved to be a highly sensitive technique and should be useful for studying the interactions between DNA and binding proteins, including DNA mismatch recognition.
Inhibition of astroglial cell proliferation by alcohols: interference with the protein kinase C‐phospholipase D signaling pathway
Authors: Kötter Katja , Jin Shenchu , Klein Jochen
Published: July 2002

Ethanol inhibits astroglial cell proliferation, an effect that may contribute to the development of alcoholic embryopathy in humans. In the present study, we investigated inhibitory effects of ethanol and butanol isomers (1‐, 2‐ and t‐butanol) on astroglial cell proliferation induced by the strongly mitogenic phorbol ester, 4ß‐phorbol‐12α,13ß‐dibutyrate (PDB). 4ß‐Phorbol‐12α,13ß‐dibutyrate (PDB) induced a 10‐fold increase of [3H]thymidine incorporation in cortical astrocytes prepared from newborn rats (EC50: 70 nM) which was blocked by Ro 31‐8220, a cell‐permeable protein kinase C (PKC) inhibitor. Ethanol blocked PDB‐induced astroglial proliferation in a concentration‐dependent manner; significant effects were already seen at 0.1% (v/v). Concomitantly, ethanol caused the formation of phosphatidylethanol (PEth) by phospholipase D (PLD) and reduced PLD‐mediated formation of phosphatidic acid (PA). The butanols also inhibited the mitogenic action of phorbol ester; the inhibitory potency of the butanols was 1‐butanol>2‐butanol>t‐butanol. The same range of potencies was observed for the inhibitory activity of the butanols towards protein kinase C activity measured in vitro. At 0.3% concentration, 1‐butanol potently suppressed the PDB‐induced formation of phosphatidic acid while 2‐ and t‐butanol were less active. Taken together, our results suggest that ethanol and 1‐butanol exert a specific inhibitory effect on PKC‐dependent astroglial cell proliferation by synergistically inhibiting PKC activity and the PLD signaling pathway.
Immortalized hypothalamic luteinizing hormone‐releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: involvement of basic fibroblast growth factor
Authors: Avola Roberto , Spina‐Purrello Vittoria , Gallo Francesco , Morale Maria C. , Marletta Nunzio , Costa Antonino , Tirolo Cataldo , Testa Nuccio , Reale Salvatore , Marchetti Bianca
Published: July 2002

Recent evidence indicates that astroglial‐derived growth factors (GFs) participate in the development of luteinizing hormone‐releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1‐1) neurons in co‐culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial‐induced acquisition of the mature LHRH phenotype by GT1‐1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin‐like growth factor I, IGF‐I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co‐culture with pure LHRH neurons. Pre‐treatment of hypothalamic astrocytes with an inactive (‘priming’) dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF‐I (10 ng/ml), EGF (10 g/ml) and insulin (10 μg/ml), inducing a 65–100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1‐1 neurons were co‐cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co‐culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH‐immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1‐1 neuron–glial co‐cultures by a bFGF‐antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.