bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of a putative Na+ /D‐glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column
Authors: Kitlar Tomas , Morrison Alison I. , Kinne Rolf , Deutscher Josef
Published: July 2002

Phlorizin, a potent inhibitor of the Na+ /D‐glucose cotransporter, was derivatised to 3‐aminophlorizin and subsequently coupled to Affi‐Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X‐100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na+ /D‐glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi‐Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na+; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.
Convenient plasmid vectors for construction of chimeric mouse/human antibodies
Authors: Kameyama Koh-zoh , Imai Kenji , Itoh Toshihiro , Taniguchi Masaru , Miura Keiji , Kurosawa Yoshikazu
Published: July 2002

Chimeric antibodies composed of mouse‐derived variable regions and human‐derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time‐consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and Vϰ genes can be identified as rearranged bands by Southern hybridization of EcoRI‐ and HindIII‐digested DNAs with JH and Jϰ probes, respectively, and such fragments can be isolated in λ‐EcoRI and λ‐HindIII vectors, respectively. We constructed two plasmids: pSV2‐HG1gpt contains human Cγ1 and Ecogpt genes, and only one EcoRI site upstream of the Cγ1 gene; pSV2‐HCϰneo contains human Cϰ and neo genes, and only one HindIII site upstream of the Cϰ gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a VϰJϰ gene are inserted into pSV2‐HG1gpt and pSV2‐HCϰneo, respectively. Both resulting plasmid DNAs are co‐transfected into SP2/0 cell, a non‐Ig‐secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.
Phospholipase C and phospholipase A2 are involved in the antiviral activity of human interferon‐α
Authors: Premecz György , Markovits Andrea , Bagi György , Farkas Tibor , Földes István
Published: July 2002

Treatment of human amniotic cells (UAC) with human interferon‐α (Hu‐IFNα) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK‐C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN‐treated cells suggest that phospholipases C and A2 are activated by Hu‐IFNα . Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN‐treated cells inhibited the antiviral activity of Hu‐IFNα in the vesicular stomatitis virus (VSV)‐UAC system used. These observations indicate that (i) activation of PK‐C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.
Induction of tumour hypoxia by a vasoactive agent A combined NMR and radiobiological study
Authors: Dunn J.F. , Frostick S. , Adams G.E. , Stratford I.J. , Howells N. , Hogan G. , Radda G.K.
Published: July 2002

The effect of hydralazine treatment on 3 murine tumours (RIF‐1, KHT and 16/C) was monitored using 31P‐NMR. Changes in the 31P‐NMR spectrum are compared with measurements of radiobiological hypoxic fraction (RHF) in the RIF‐1 and KHT. Hydralazine is known to reduce temporarily blood flow in experimental tumours, and thus cause a transient increase in the RHF to 100% (in RIF‐1 and KHT). This correlates with a decline in energy status as measured by 31P‐NMR (i.e. there was an increase in Pi in all three tumours). Time‐course data from the RIF‐1 and KHT tumours show that maintenance of anaesthesia prolongs the hypoxia induced by hydralazine.
Alzheimer's disease: Microtubule‐associated proteins 2 (MAP 2) are not components of paired helical filaments
Authors: Rosemblatt M. , Fellous A. , Mazie J.C. , Delacourte A. , Defossez A.
Published: July 2002

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill‐defined, a number of microtubule‐associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule‐associated protein MAP 2 which does not cross‐react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule‐associated protein τ shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the τ proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti‐MAP 2 antiserum suggests a non‐specific binding of MAP 2 to the PHF during the process of NFT formation.
A novel, specific binding protein assay for quantitation of intracellular inositol 1,3,4,5‐tetrakisphosphate (InsP4) using a high‐affinity InsP4 receptor from cerebellum
Authors: Donié Frédéric , Reiser Georg
Published: July 2002

A membrane preparation from porcine cerebellum displays high‐affinity binding sites for [3H]inositol 1,3,4,5‐tetrakisphosphate ([3H]InsP4) with a dissociation constant (K
d) of 1.0 nM and a density of 220 fmol/mg protein. Specific binding was maximal in the presence of 25 mM phosphate and at pH 5.0. The receptor site was specific for InsP4, since Ins(1,3,4,5,6)P5 and Ins(1,4,5,6)P4 displaced binding of InsP4 with EC50 values of 0.2 and 0.3 μM, respectively. Ins(1,4,5)P3 and other inositol phosphates were less effective. Using this InsP4 receptor, an assay for measuring tissue content of InsP4 was developed. The detection limit of the assay was 0.1 pmol. In the same tissue samples the amount of Ins(1,4,5)P3 was determined in parallel with a similar assay using a binding protein preparation from beef liver.
Parallel stranded DNA with AT base pairing
Authors: Shchyolkina A.K. , Lysov Yu.P. , Il'ichova I.A. , Chernyi A.A. , Golova Yu.B. , Chernov B.K. , Gottikh B.P. , Florentiev V.L.
Published: July 2002

The concentration and temperature dependences of the UV and CD spectra of the oligonucleotide 3′‐d(ApTpApTpApTpApTpApTp)‐O(CH2)6O‐5′‐d(pApTpApTpApTpApTpApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NACl were studied. At less than 10−6 M, the eicosamer was shown to form in solution a hairpin with parallel orientation of chains (parallel hairpin). From thermal denaturation profiles [A
260(T)] the thermodynamic parameters, ΔH°, ΔS° and T
m for parallel hairpin formation were calculated to be −90±8 kJ/mol. −300±20 J · mol−1 · K−1 and 40.5°C, respectively. The CD spectra of the parallel double helix differed from those of B‐form DNA and had characteristic features: decreasing magnitude of the positive maximum at 265 nm and a negative peak at 285 nm.
Poly(ADP‐ribose) polymerase activity is inhibited by 2',5'‐oligoadenylates in mouse L‐cells
Authors: Buonamassa Daniela Tornese , Malanga Maria , Coccia Eliana M. , Romeo Giovanna , Affabris Elisabetta , Farina Benedetta , Suzuki Hisanori
Published: July 2002

Down regulation of poly(ADP‐ribose)polymerase (ADPRP) activity was observed in mouse LW‐cells after treatment with 2'‐5'oligoadenylates or with fibroblast interferon and poly(rI).poly(rC). The poly(rI).poly(rC)‐induced inhibition of the enzymatic activity correlates with the observed increase of endogenous 2',5'‐oligoadenylate cores which were reported to be potent inhibitors of ADPRP in vitro.
A repeated decapeptide motif in the C‐terminal domain of the ribosomal RNA methyltransferase from the erythromycin producer Saccharopolyspora erythraea
Authors: Dhillon Namrita , Leadlay Peter F.
Published: July 2002

Re‐analysis of the primary structure of the ribosomal RNA N‐methyltransferase that confers self‐resistance on the erythromycin‐producing bacterium Saccharopolyspora erythraea has confirmed the presence of a C‐terminal domain containing extensive repeat sequences. Nine tandem repeats can be discerned, with a decapeptide consensus sequence GGRx(H/R)GDRRT, although no single residue is wholly invariant. This highly polar, potentially flexible domain, which is predicted to adopt either a random coil or a structure with β turns, has a counterpart in the erythromycin methyltransferase of an erythromycin‐producing species of Arthrobacter. It also significantly resembles a portion of the C‐tenninal region of the eukaryotic protein nucleolin, which is unusually rich in dimethylarginine and glycine, and which is also predicted to behave as a random coil in solution. This resemblance, despite the very different roles of these proteins in ribosome biogenesis, strengthens the idea that in both rRNA methyltransferases and nucleolin these C‐tenninal sequences might contribute to rRNA binding.
2D COSY 1H NMR: a new tool for studying in situ brain metabolism in the living animal
Authors: Barrére Bertrand , Peres Michel , Gillet Brigitte , Mergui Simone , Beloeil Jean-Claude , Seylaz Jacques
Published: July 2002

2D COSY 1H NMR with surface coil has been used to resolve and assign cerebral metabolites which had previously been detected but could not be resolved or assigned in situ in the living animal by conventional ID 1H NMR. A wide range of cerebral metabolites, including alanine, N‐acetyl aspartate, aspartate, choline derivatives, creatine/phosphocreatine pool, GABA, glucose, glutamate/glutamine pool, inositol, lactate and taurine were simultaneously resolved and assigned in situ in the whole animal using the 2D COSY correlation graphs. Global irreversible ischemia caused the appearance and the disappearance of cross‐peaks in the 2D COSY 1H NMR map, corresponding to increases in alanine, GABA and lactate and glucose depletion.
Amino‐terminal sequence of the 21 kDa apoprotein of a minor light‐ harvesting pigment‐protein complex of the Photosystem II antenna (LHC IId/CP24)
Authors: Morishige Daryl T. , Anandan Shivanthi , Jaing James T. , Thornber J.Philip
Published: July 2002

The 21 kDa apoprotein of LHC IId, a minor light‐harvesting antenna component of Photosystem II, has been isolated and subjected to N‐tenninal protein sequencing. A sequence of 66 residues was obtained which contains regions of considerable homology to both those reported for LHC II and LHC I, but which is obviously distinct from them. The proposed occurrence of an identical 21 kDa LHC subunit in both photosystems I and II is shown to be incorrect.
In vitro inhibition of HIV‐1 proteinase by cerulenin
Authors: Moelling Karin , Schulze Thomas , Knoop Marie-Therés , Kay John , Jupp Raymond , Nicolaou George , Pearl Laurence H.
Published: July 2002

Retroviruses encode proteinases necessary for the proteolytic processing of the viral gag and gag‐pol precursor proteins. These enzymes have been shown to be structurally and functionally related to aspartyl proteinases such as pepsin and renin. Cerulenin is a naturally occurring antibiotic, commonly used as an inhibitor of fatty acid synthesis. Cerulenin has been observed to inhibit production of Rous sarcoma virus and murine leukaemia virus by infected cells, possibly by interfering with proteolytic processing of viral precursor proteins. We show here that cerulenin inhibits the action of the HIV‐1 proteinase in vitro, using 3 substrates: a synthetic heptapeptide (SQNYPIV) which corresponds to the sequence at the HIV‐1 gag
junction, a bacterially expressed gag precursor, and purified 66 kDa reverse transcriptase. Inhibition of cleavage by HIV‐1 proteinase required preincubation with cerulenin. Cerulenin also inactivates endothiapepsin, a well‐characterised fungal aspartyl proteinase, suggesting that the action of cerulenin is a function of the common active site structure of the retroviral and aspartic proteinases. Molecular modelling suggests that cerulenin possesses several of the necessary structural features of an inhibitor of aspartyl proteinases and retroviral proteinases. Although cerulenin itself is cytotoxic and inappropriate for clinical use, it may provide leads for the rational design of inhibitors of the HIV proteinase which could have application in the chemotherapy of AIDS.
Large scale preparation of homogeneous bacteriorhodopsin
Authors: Lorber Bernard , DeLucas Larry J.
Published: July 2002

Homogeneous bacteriorhodopsin was obtained preparatively (100 mg batches) from purple membrane of Halobacterium halobium cells. The homogeneity of the protein was considerably affected by variations in the growth conditions of the bacteria. Fully maturated bacteriorhodopsin having a blocked N‐terminus and a homogeneous C‐terminus, was reproducibly obtained when cells were grown in a sufficiently aerated medium.
Ouabain‐insensitive, Na‐ATPase activity in pure suspensions of rat kidney proximal tubules
Authors: Marín Reinaldo , Gómez Daniela C. , Rodríguez Gloria A. , Proverbio Teresa , Proverbio Fulgencio
Published: July 2002

The present work was undertaken to evaluate the distribution of the Na‐ATPase activity in the different components of the rat kidney cortex. Suspensions of glomeruli, proximal and distal tubules were prepared following a collagenase digestion of outermost kidney cortex slices and a separation on a Percoll gradient. It was found that the Na‐ATPase activity is higher in the fraction enriched in proximal tubules. The fraction enriched in glomeruli and in distal tubules show also a Na‐ATPase activity, but it is lower.
Specific binding of placental acidic isoferritin to cells of the T‐cell line HD‐MAR
Authors: Konijn A.M. , Meyron-Holtz E.G. , Levy R. , Ben-Bassat H. , Matzner Y.
Published: July 2002

Acidic placental isoferritin inhibited the blastogenic response of peripheral human lymphocytes to T‐cell activating lectins. We measured specific binding of radioiodinated placental isoferritin to cells of the T‐cell line HD‐MAR and found specific high‐affinity binding. Binding was faster and more ferritin was bound at 37°C than at 4°C. Displacement experiments indicated that most of the binding occurred at the cell surface. Acidic placental ferritin and isolated H‐type ferritin subunits but not isolated L‐type subunits, competed for the binding. Scatchard plot analysis showed characteristics of a single binding species with a dissociation constant (K
d) of 1.3–4.4 × 10−11 M. The results suggest the presence of receptors for acidic isoferritin on T‐lymphocytes and thus, a regulatory role for the acidic ferritin H‐type subunit in T‐cell function.
Engineering thermostability in archaebacterial glyceraldehyde‐3‐phosphate dehydrogenase Hints for the important role of interdomain contacts in stabilizing protein conformation
Authors: Biro Jutta , Fabry Stefan , Dietmaier Wolfgang , Bogedain Christoph , Hensel Reinhard
Published: July 2002

Construction of hybrid enzymes between the glyceraldehyde‐3‐phosphate dehydrogenases from the mesophilic Methanobacterium bryantil and the thermophilic Methanothermus fervidus by recombinant DNA techniques revealed that a short C‐terminal fragment of the Mt.fervidus enzyme contributes largely to its thermostability. This C‐terminal region appears to be homologous to the α6‐helix of cubacterial and eukaryotie glyceraldehyde‐3‐phosphate dehydrogenases which is involved in the contacts between the two domains of the enzyme subunit. Site‐directed mutagenesis experiments indicate that hydrophobic interaction play an important role in these contacts.
Subcellular distribution of α, and β subunit proteins of Ca2+ /calmodulin‐dependent protein kinase II expressed in Chinese hamster ovary cells
Authors: Yamauchi Takashi , Sekihara Shun-ichi , Ohsako Shunji
Published: July 2002

When two cDNAs respectively encoding the entire coding regions of α and β subunits of Ca2+ /calmodulin‐dependent protein kinase II (CaM kinase II) were introduced into Chinese hamster ovary cells, the expressed α and β subunits were differently associated with subcellular structure. Although α subunit was loosely associated with subcellular structure, about 80% of CaM kinase II acitivity of α subunit was found in soluble fraction. More than 50% of the β subunit bound to the membrane, and the remainder was soluble but was loosely associated with subcellular structure. The relative rate of phosphorylation for substrate proteins of the β subunit bound to membrane was significantly different from that of the soluble form.
Biochemical evidence for histidine oxidation in photosystem II depleted of the Mn‐cluster for O2‐evolution
Authors: Ono Taka-aki , Inoue Yorinao
Published: July 2002

Flash excitation of Tris‐treated photosystem II (PSII) membranes generated a thermoluminescene band peaking ‐25°C. Indicating that a positively charged oxidizing equavalent is stably accumulated on donor side of PSII even in the Mn‐cluster. This oxidizing equivalent was sensitive to low concentrations of exogenous Mn???, and its stable accumulation was reversibly inhibited by diethylpyrocarbonate treatment that modifies histidine residues with high specificity. It was inferred that there is a photooxidizable histidine residue(s) on donor side of PSII and it provides a redox‐active ligand for Mn
Induction of morphological change by tyrosine kinase inhibitors in Rous sarcoma virus‐transformed rat kidney cells
Authors: Umezawa Kazuo , Tanaka Karo , Hori Takashi , Abe Satoko , Sekizawa Ryuichi , Imoto Masaya
Published: July 2002

Erbstatin and methyl 2,5‐dihydroxycinnamate, related tyrosine kinase inhibitors, induced a morphological change in temperature‐sensitive Rous sarcoma virus‐transformed rat kidney (RSV15·NRK) that brought the cells close to the morphology of their normal counterpart. Erbstatin did not change the morphology of normal or Kirsten sarcoma virus‐transformed rat kidney cells. Erbstatin also inhibited morphological transformation of RSV15·NRK cells induced by a shifting in temperature. Actin stress fibres were observed only in normal cells and not in transformed cells. Erbstatin induced stress fibre organization in transformed cells. Erbstatin and methyl 2.5‐dihydroxycinnamate increased fibronectin gene expression in RSV‐transformed cells. Thus, tyrosine kinase inhibitors induced normal phenotypes specifically in v‐sre‐expressing cells.
Complete nucleotide sequence of the Mycobacterium leprae 23 S and 5 S rRNA genes plus flanking regions and their potential in designing diagnostic oligonucleotide probes
Authors: Liesack W. , Sela S. , Bercovier H. , Pitulle C. , Stackebrandt E.
Published: July 2002

The complete nucleotide sequences of the Mycobacterium leprae 23 S and 5 S rRNA genes and their flanking regions are presented. As compared to other eubacterial homologous molecules the 23 S rDNA exhibits two insertions. A 16 nucleotide long insertion is almost unique to members of the genus Mycobacterium, while the second represents an extended version of helix 54. The potential or both insertions to serve as target for diagnostic oligonucleotide probes was proven by comparative sequence analysis of 23 S rRNA or several Mycobacterium species and by dot blot hybridization. In addition, a 19‐mer oligonucleotide probe is described, which can be considered genus Mycobacterium‐specific.
SRH1 protein, the yeast homologue of the 54 kDa subunit of signal recognition particle, is involved in ER translocation of secretory proteins
Authors: Amaya Yoshihiro , Nakano Akihiko
Published: July 2002

The function of the SRH1 product, the yeast homologue of the 54 kDa subunit of the mammalian signal recognition particle, has been analyzed using a galactose dependent mutant of the gene. SRH1 has been placed under control of the GALI promoter and introduced into a haploid cell that had its chromosomal SRH1 copy disrupted. This mutant grows normally on galactose medium but slows down the growth about 10 h after transfer to glucose medium. At the same time, precursor forms of secretory proteins, α‐mating factor and invertase, accumulate in the cells. This result indicates that the SRH1 product is involved in translocation of precursors of secretory proteins across the endoplasmic reticulum membrane in yeast cells.
Growth inhibition of lysozyme crystals at high hydrostatic pressure
Authors: Groß Michael , Jaenicke Rainer
Published: July 2002

The influence of high hydrostatic pressures on the crystallization of hen egg‐white lysozyme has been determined by growing lysozyme crystals with a capillary technique adapted to high pressure conditions and monitoring the residual protein concentration remaining in the supernatant. Pressure is found to reduce the rate of crystallization and to enhance the solubility of lysozyme in 1 M NaCl, pH 4.66. The volume change calculated from the latter result amounts to 12.5 ml/mol. This value is too small to be detectable by comparison of total molar volumes.
Proton NMR investigation of the reconstitution of equine myoglobin with hemin dicyanide Evidence for late formation of the proximal His93F8‐iron bond
Authors: Yee Sidney , Peyton David H.
Published: July 2002

Reconstitution of apoequine myoglobin (apoEqMb) with hemin dicyanide (FePPIX(CN)2) was monitored by 1H NMR spectroscopy to gain information about the sequence of events leading to metEqMbCN. At least one step in the pathway is slow enough to allow us to follow the time‐dependence of formation of the product, a mixture of heme‐insertion isomers characterized by others (Jue, T., Krishnamoorthi, R. and La Mar, G.N. (1983) J. Am. Chem. Soc. 105, 5701–5703; Lecomte, J.T.J., Johnson, R.D. and La Mar, G.N. (1985) Biochim. Biophys. Acta 829, 268–274). However, in contrast to all previously reported Mb‐FePPIX reconstitutions, we find that the initial ratio of heme‐insertion isomers is not 1:1. This mixture is, instead, found to be enhanced in the isomer which dominates at equilibrium. This is taken as evidence for a ‘[FePPIX(CN)2·EqMb]’ intermediate which lacks the proximal His93F8‐Fe bond and which proceeds quickly toward an equilibrium ratio of heme‐insertion isomers. Therefore the heme‐insertion isomer ratio is frozen only when the proximal His93F8‐Fe bond is formed. The difference in this ratio of heme‐insertion isomers between EqMb(4,5:1) and SwMb(2.5:1) likely reflects the amino acid substitution: Lys→Arg45CD3(EqMb→SwMb).
Specific binding of phenolic glycolipid antigens from Mycobacterium bovis BCG with antibodies
Authors: Vercellone Alain , Riviere Michel , Fournié Jean-Jacques , Puzo Germain
Published: July 2002

We studied the molecular binding specificity of two rabbit polyclonal sera generated against phenolic glycolipid antigens namely PheG1 B and PheG1 B‐3 from Mycobacterium bovis BCG. PheG1 B is the well‐known mycoside B (2‐O‐Me‐α‐l‐Rhap 1 → aglycone), while PheG1 B‐3 is a recently found glycolipid (α‐l‐Rhap‐(1→3)‐2‐O‐Me‐α‐l‐Rhap 1 → aglycone). The interaction specificity was mainly explained in terms or the cavity volume of the antibodies paratope. The anti‐PheG1 B antibodies paratope fits the 2‐O‐Me‐α‐l‐Rhap ligand, while that of anti‐PheG1 B‐3 binds the disaccharide moiety of PheG1 B‐3, and, with a higher affinity, the monosaccharidic unit localized at the non‐reducing end. The B‐3 antigen affinity is higher than that of antigen B for their homologous antibodies. This can be explained by the fact that the antibodies against phenolic glycolipid B‐3 bind optimally to two sequential glycosyl residues suggesting the presence of two subsites. The immunoglobulin subsite with the major affinity binds the monosaccharidic unit localized at the non‐reducing end.
Altered protein folding may be the molecular basis of most cases of cystic fibrosis
Authors: Thomas Philip J. , Ko Young H. , Pedersen Peter L.
Published: July 2002

Experiments have demonstrated that the cystic fibrosis transmembrane conductance regulator protein (CFTR), containing the most common cystic fibrosis (CF)‐causing mutation (ΔF508), reaches the plasma membrane in reduced amounts. Studies of a peptide model of CFTR indicate that the ΔF508 mutated region is more sensitive to denaturating conditions. This paper proposes that altered protein folding accounts for these findings, and, thus, most cases of CF. Significantly, the hypothesis makes specific predictions about the effect of stabilizing conditions on mutant CFTR, and, further, suggests a new class of pharmaceuticals that may prove effective in the treatment of this important genetic disease.
A GC box in the bidirectional promoter is essential for expression of the human dihydrofolate reductase and mismatch repair protein 1 genes
Authors: Fujii Hiroyuki , Shinya Eiji , Shimada Takashi
Published: July 2002

The human dehydrofolate reductase and mismatch repair protein 1 genes are organized in a head‐to‐head configuration separated by an 88 base‐pair segment and directed by a bidirectional promoter. In vivo transient assays of the site directed mutant promoters using firefly luciferase as a reporter showed that an AT‐rich sequence, ACAAATA, in the GC‐rich promoter sequence is not required for transcription. However, two out of four GC boxes were shown to function as bidirectional positive regulatory elements. Among them, a GC box at the midpoint of the region between the two initiation sites is essential for supporting minimal bidirectional activity.
Procathepsin D cannot autoactivate to cathepsin D at acid pH
Authors: Larsen Lotte B. , Boisen Anni , Petersen Torben E.
Published: July 2002

The amino acid sequence of the propart of bovine procathepsin D was determined at the protein level. Incubation of the isolated procathepsin D at pH 3.5–5.0 for 30–120 min leads to a 2 kDa reduction in its molecular mass, as seen by SDS‐PAGE. The activation product is pseudocathepsin D and is the result of a proteolytic cleavage between LeuP26 and IleP27 in the propart. Incubation at pH 5.0 for 20 h of either procathepsin D or pseudocathepsin D results in both cases in approximately equal amounts of pseudocathepsin D and a further processed intermediate, nine amino acids shorter than pseudocathepsin D. No reaction products corresponding to cathepsin D with a mature amino terminus were observed, showing that autoproteolysis alone cannot generate the mature form found in the lysosomes.
Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes
Authors: Racke Frederick K. , Hammerland Lance G. , Dubyak George R. , Nemeth Edward F.
Published: July 2002

Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca2+. To test this hypothesis, Xenopus leavis oocytes were injected with poly(A)+‐enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca2+ or other polycations. Cytosolic Ca2+ concentrations were measured indirectly by recording Cl− currents through the endogenous, cytosolic Ca2+‐activated Cl− channel. Increasing the concentration of extracellular Ca2+ (from 0.7 to 5 mM) or Mg2+ (from 0.8 to 10 mM) elicited oscillatory increases in the CT current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL‐60 cells or rat liver. Responses elicited by extracellular Mg2+ persisted when extracellular Ca2+ was reduced to low micromolar levels. La3+, Gd3+, or neomycin B also evoked oscillatory increases in the Cl− current in oocytes under conditions of low extracellular Ca2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca2+ receptor protein(s) on parathyroid cells that regulates cellular function.
Synthesis of functional GABAA receptors in stable insect cell lines
Authors: Joyce Kirsti A. , Atkinson Allan E. , Bermudez Isabel , Beadle David J. , King Linda A.
Published: July 2002

We have synthesised the β1‐subunit of the bovine GABAA receptor in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovirus immediate early (IE‐1) gene promoter. Transformed cells were obtained by co‐transfection of the insect cells with pIEK 1 .GRβ1, encoding the β1 subunit cDNA, and pIEK1 .neo, encoding the neomycin resistance gene. G‐418‐resistant clones were selected and expanded into continuous cell lines synthesising functional, GABA‐gated, homo‐oligomeric chloride channels. These cell lines had significant advantages over the transient baculovirus expression system for the characterisation of receptors using electrophysiological recording techniques.
Interaction of lactogenic hormones with purified recombinant extracellular domain of rabbit prolactin receptor expressed in insect cells
Authors: Gertler Arieh , Petridou Barbara , Kriwi Gwen G. , Djiane Jean
Published: July 2002

The extracellular domain of rabbit prolactin receptor (rbPRLR‐ECD) expressed in an insect/baculovirus expression system was purified by affinity chromatography on immobilized PRL followed by gel filtration. The purified protein was over 90% homogeneous as indicated by SDS‐PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Its molecular mass determined by SDS‐PAGE was 32 kDa, and by gel filtration, 27 kDa. Both values are higher than the 22.8 kDa deduced from the cDNA sequence, indicating extensive glycosylation. The K
a value for interaction with ovine (o) PRL was 25.4 nM−1, but even at high rbPRLR‐ECD:hormone molar ratios, the stoichiometry of interaction with oPRL or human growth hormone indicated formation of only 1:1 complexes, in contrast to human growth hormone (hGH)‐ECD which forms 2:1 complexes with hGH.
Mouse monoclonal antibodies that specifically recognize an amino‐terminal epitope of p56lck protein tyrosine kinase
Authors: Stieger Martin , Flint Nicholas A. , Jacot-Guillermod Hubert , Burn Paul
Published: July 2002

p56lck is a T cell‐specific protein tyrosine kinase of the src family of proto‐oncogenes which has been implicated in T cell signal transduction. Here we describe the production of mouse monoclonal antibodies directed against recombinant human p56lck purified from an E. coli expression system. The antibodies were characterized by ELISA, RIA and immunoprecipitation of p56lck from T cell lysates. A specific epitope was revealed at the aminoterminus of the p56lck molecule by using Western blotting of deletion mutants and distinct domains of p56lck expressed in E. coli. Potential applications of the results obtained are discussed.
Polysialylated neural cell adhesion molecule expression by neurons and astrolial molecule in the rat dentate gyrus declines dramatically with increasing age
Authors: Fox G.B. , Kennedy N. , Regan C.M.
Published: July 2002

The expression of polysialylated neurons in the dentate gyrus of the hippocampal formation of young (postnatal day 40), mature (postnatal day 80) and aged (postnatal day 540) male Wistar rats has been investigated by immunohistochemical techniques employing a monoclonal antibody specific for neural cell adhesion molecule‐linked α2,8 polysialic acid. A strong immunoreactivity was found on the cell bodies, dendrites and axons of granule‐like neuronal cells at the border between the hilar region and the granule cell layer of the young rat. In the mature animal the number of immunoreactive neurons declined dramatically and were virtually absent in the aged group. Using an alternative fixation procedure, glial fibrillary acidic protein‐positive and polysialylated astroglia processes were found in close proximity to the dendrites of the polysialylated granule‐like cells. The number of astroglial processes traversing the granule cell layer showed a similar age‐dependent decline to that observed with the polysialylated neurons. Glial fibrillary acidic protein‐positive and polysialylated stellate astroglia were present throughout the hippocampal formation, but did not show the marked age‐dependent decline observed with the astroglial processes in the granule cell layer.

The neuronal dendrites and astroglial processes exhibited a strict numerical ratio in the young and mature animal and, in double immunofluorescence studies with anti‐polysialic acid and anti‐glial fibrillary acidic protein, the astroglial processes exhibited apparent points of cell and/or dendritic contact. These findings suggest that loss of polysialylated astroglial processes precedes the decline in polysialylated dentate neurons.
Atypical antipsychotic drugs in dysthymia: placebo controlled studies of amisulpride versus imipramine, versus amineptine
Authors: Boyer P , Lecrubier Y
Published: July 2002

In order to assess the therapeutic efficacy of amisulpride in dysthymic disorder, two randomised double-blind trials were conducted in France. Both studies included outpatients meeting DSM-III-R criteria for primary dysthymia. Cases of double depressed patients with a mild or moderate major depressive episode superimposed to dysthymia were also eligible. The first study of a three-month duration compared amineptine (200 mg/day) to amisulpride (50 mg/day) and placebo. The second study of a six-month duration compared the therapeutic effects of imipramine (100 mg/day) to amisulpride (50 mg/day) and placebo. Results of the intention to treat analysis and of the end-point analysis were compelling and very similar: significant differences were demonstrated for all primary criteria between amisulpride and placebo and between imipramine and placebo but not between amisulpride and imipramine. For both primary criteria and the responder rate (CGI), statistically significant differences were evidenced between amisulpride and placebo and amineptine and placebo. The same differences were evidenced for SANS subscores. Beneficial effects of low doses of amisulpride in chronic depressive condition can therefore be discussed.
A CDE/CHR‐like element mediates repression of transcription of the mouse RB2 (p130) gene
Authors: Fajas Lluis , Le Cam Laurent , Polanowska Jolanta , Fabbrizio Eric , Servant Nadège , Philips Alexandre , Carnac Gilles , Sardet Claude
Published: July 2002

The bipartite repressor elements, termed cell cycle‐dependent element (CDE)/cell cycle regulatory element (CCRE)‐cell cycle homology region (CHR) control the growth‐dependent transcription of the cyclin A, cdc25C, cdc2 genes. Here, we have identified a functional element displaying the signature of the CDE–CHR in the promoter of the mouse RB2 (p130) gene, encoding the retinoblastoma protein family (pRB)‐related protein p130. This element locates close to the major transcription start site where it makes major groove contacts with proteins that can be detected in a cellular context using in vivo genomic footprinting techniques. Inactivation of either the CDE or CHR sequence strongly up‐regulates the p130 promoter activity in exponentially growing cells, a situation where endogenous p130 gene expression is almost undetectable. Electrophoretic mobility shift assays suggest that two different protein complexes bind independently to the p130 CDE and CHR elements, and that the protein(s) bound to the CDE might be related to those bound on cyclin A and cdc2 promoters.
Evidence for a pool of coronin in mammalian cells that is sensitive to PI 3‐kinase
Authors: Didichenko S.A. , Segal A.W. , Thelen M.
Published: July 2002

Coronin, a 57 kDa actin binding protein elutes with an apparent molecular mass of 400–600 kDa from gel filtration columns. This fraction is not unrelated to the reported 200 kDa complex where coronin is associated with phox proteins of the NADPH‐oxidase. Phosphatidylinositol 3‐kinase (PI 3‐kinase) solubilizes coronin from the 400–600 kDa complex, thus constitutive active PI 3‐kinase is sufficient to disrupt the complex, whereas wortmannin stabilizes it. Conversely, the phox protein associated pool of coronin is PI 3‐kinase independent. During phagocytosis coronin is recruited together with PI 3‐kinase to membranes of nascent and early phagosomes co‐localizing with the actin cytoskeleton, confirming that coronin contributes to phagocytosis.
Identification of the main ubiquitination site in human erythroid α‐spectrin
Authors: Galluzzi Luca , Paiardini Mirko , Lecomte Marie C , Magnani Mauro
Published: July 2002

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid α‐spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of α‐spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site‐directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in α‐spectrin and may be present in several other proteins.
Osteopontin involvement in integrin‐mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells
Authors: Liu Yin-Kun , Uemura Toshimasa , Nemoto Atsuko , Yabe Tomoko , Fujii Noriko , Ushida Takashi , Tateishi Tetsuya
Published: July 2002

To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106‐6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts.
Preparation of basal cell membranes for scanning probe microscopy
Authors: Ziegler U , Vinckier A , Kernen P , Zeisel D , Biber J , Semenza G , Murer H , Groscurth P
Published: July 2002

Scanning probe microscopy has the potential for investigating membranes in a physiological environment. We prepared with a lysis‐squirting protocol basal cell membranes, that are suitable for scanning probe microscopy. Investigations using atomic force microscopy under liquid revealed cellular filaments which correlated perfectly with fluorescently stained actin filaments. Globular structures with a diameter as little as 10 nm could be resolved by stripping cytoplasmic components from the membranes. Therefore, cytoplasmic sides of supported basal cell membranes prove useful to gain high resolution with scanning probe microscopy in studies of plasma membrane associated structures and processes under buffer solution.
The cleavage site specificity of human prostate specific antigen for insulin‐like growth factor binding protein‐3
Authors: Okabe Eriko , Kajihara Jun-ichi , Usami Yoshiko , Hirano Kazuyuki
Published: July 2002

The cleavage site of human insulin‐like growth factor binding protein‐3 by urinary prostate specific antigen was examined. human insulin‐like growth factor binding protein‐3 was incubated with urinary prostate specific antigen at 37°C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N‐terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr‐159. The synthetic peptide including Tyr‐159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin‐like growth factor binding protein‐3 is specifically cleaved at Tyr‐159 by prostate specific antigen. human insulin‐like growth factor binding protein‐3 was previously reported to be cleaved at five sites including Arg‐97, Arg‐132, Tyr‐159, Phe‐173 and Arg‐179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin‐like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin‐like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin‐like growth factor binding protein‐3.
Phosphorylation of p67phox in the neutrophil occurs in the cytosol and is independent of p47phox
Authors: Forbes Louisa V. , Moss Stephen J. , Segal Anthony W.
Published: July 2002

p67phox and p47phox are phosphorylated in the course of stimulation of the NADPH oxidase in neutrophils. Isolated neutrophil cytosol can phosphorylate both of these proteins in vitro. Phosphoamino acid analysis showed that isolated membranes can tyrosine‐phosphorylate p67phox in vitro. Further experiments with anti‐phosphotyrosine antibodies did not support a role for tyrosine phosphorylation of p67phox in the cell. A phosphopeptide analysis showed that the phosphorylation of p67phox is unchanged in the absence of p47phox. These results further characterise the phosphorylation of p67phox and provide evidence that this is a cytosolic event independent of interaction with p47phox and the membrane.
Pregnancy during adolescence: Wanted vs. unwanted
Authors: Pinto e Silva J.L.
Published: July 2002

Pregnancy among adolescents is an important problem in most developing countries. The phenomenon in Brazil seems numerically stable for the age group from 15 to 19 years old, with a trend of increase in the first segment of adolescence, below 15. The majority of pregnancies are unwanted, with medical, psychological and mainly social repercussions. The disadvantages are clearer for multipara adolescents. The main immediate consequences of an unwanted pregnancy are: induced abortion, lack of prenatal care, personal and family disruption, adoption and abandonment. Some intervention policies are suggested for the reduction of this phenomenon through programs of sexual education, services for special care of adolescents, access to orientation and contraceptive methods, and support for the pregnancies to be carried on until term.
Pushing the frontiers of science — the Population Council's Microbicide Basic Science Network on vaginal microbicide research
Authors: Johansson E.D.B. , Maguire R.A. , Phillips D.M.
Published: July 2002

Microbicides are the new frontier of products for the prevention of sexually transmitted infections (STIs). Twelve years ago, scientists realized that existing spermicides had some anti‐microbial activity and perhaps could be improved or reformulated with new compounds to provide a complete barrier against STIs. However, the development and successful marketing of an effective, non‐toxic, convenient and affordable vaginal microbicide that women can use on a long‐term basis hinges on a close collaboration between research institutions and the pharmaceutical industry. The Population Council has recently taken the first step in instituting a multifaceted strategy for the development of a microbicide by establishing the Microbicide Basic Science Network, comprising of scientists with diverse backgrounds and expertize.
Developmental pattern of plasminogen activator activity in chick optic lobe
Authors: Pereyra‐Alfonso S. , Scicolone G. , Ferrán J.L. , Saavedra J.Pecci , Flores V.
Published: July 2002

Plasminogen activators are serine proteases which play a key role in morphogenesis and tissue remodelling. Two different molecular types, tissue‐type and urokinase‐type, were identified and they were postulated to play a role in neural development. The developing chick optic lobe plays a central role in processing visual information. In previous studies we demonstrated the occurrence of high levels of plasminogen activator activity in this model. The aim of the present paper is to study the temporal pattern of expression of this activity and characterize the type of plasminogen activator expressed in the developing optic lobe.

Using soluble fractions derived by ultracentrifugation from Triton X‐100‐treated membrane fractions we measured the protease activity with a radial fibrinolytic assay. Employing different inhibitors of fibrinolytic activity and a zymographic assay, we showed that the developing optic lobe expresses only one type of plasminogen activator which corresponds to an urokinase‐type of 70 kDa. Our results indicate that peaks of protease activity temporally correlate with massive neuronal migration, neurite outgrowth and synapse formation and maturation. This suggests that a plasminogen activator could play a role in these developmental events. This consistent pattern of variability strongly suggests that it is developmentally regulated and, if so, it could be a reliable parameter to study neural plastic changes induced by modifications in the environmental stimulation.
11C‐PK11195 PET–based molecular study of microglia activation in SOD1 amyotrophic lateral sclerosis
Authors: Tondo Giacomo , Iaccarino Leonardo , Cerami Chiara , Vanoli Giovanna Emilia , Presotto Luca , Masiello Valeria , Coliva Angela , Salvi Fabrizio , Bartolomei Ilaria , Mosca Lorena , Lunetta Christian , Perani Daniela
Published: August 2020

Objective

Neuroinflammation is considered a key driver for neurodegeneration in several neurological diseases, including amyotrophic lateral sclerosis (ALS). SOD1 mutations cause about 20% of familial ALS, and related pathology might generate microglial activation triggering neurodegeneration. 11C‐PK11195 is the prototypical and most validated PET radiotracer, targeting the 18‐kDa translocator protein which is overexpressed in activated microglia. In this study, we investigated microglia activation in asymptomatic (ASYM) and symptomatic (SYM) SOD1 mutated carriers, by using 11C‐PK11195 and PET imaging.

Methods

We included 20 subjects: 4 ASYM‐carriers, neurologically normal, 6 SYM‐carriers with probable ALS, and 10 healthy controls. A receptor parametric mapping procedure estimated 11C‐PK11195 binding potentials and voxel‐wise statistical comparisons were performed at group and single‐subject levels.

Results

Both the SYM‐ and ASYM‐carriers showed significant microglia activation in cortical and subcortical structures, with variable patterns at individual level. Clusters of activation were present in occipital and temporal regions, cerebellum, thalamus, and medulla oblongata. Notably, SYM‐carriers showed microglia activation also in supplementary and primary motor cortices and in the somatosensory regions.

Interpretation

In vivo neuroinflammation occurred in all SOD1 mutated cases since the presymptomatic stages, as shown by a significant cortical and subcortical microglia activation. The involvement of sensorimotor cortex became evident at the symptomatic disease stage. Although our data indicate the role of in vivo PET imaging for assessing resident microglia in the investigation of SOD1‐ALS pathophysiology, further studies are needed to clarify the temporal and spatial dynamics of microglia activation and its relationship with neurodegeneration.