bluuebunny/crossref_metadata_2025 huggingface dataset:
Site‐selective 8‐chloroadenosine 3′,5′‐cyclic monophosphate inhibits transformation and transforming growth factor α production in Ki‐ras‐transformed rat fibroblasts
Authors: Tortora Giampaolo , Ciardiello Fortunato , Ally Shamsia , Clair Timothy , Salomon David S. , Cho-Chung Yoon Sang
Published: July 2002

A site‐selective cAMP analog, 8‐chloroadenosine 3′,5′‐cyclic monophosphate (8‐Cl‐cAMP), was demonstrated to be a potent inhibitor of both the monolayer and soft agar growth of normal rat kidney (NRK) fibroblasts that had been transformed with the v‐Ki‐ras oncogene or treated with transforming growth factor α (TGFα). The growth inhibition was dose dependent and reversible and was accompanied by reversion of the transformed phenotype, suppression of TGFα production, and a decrease in p21ras protein levels. These effects of 8‐Cl‐cAMP were linked to the cAMP analog's selective modulation of the type I and type II cAMP‐dependent protein kinase regulatory subunits, RI and RII, present in Ki‐ras‐transformed and TGFα‐treated NRK cells.
Characterization of the two rRNA gene operons present in Thiobacillus ferrooxidans
Authors: Salazar Oriana , Takamiya Mónica , Orellana Omar
Published: July 2002

The organization of rRNA genes from the autotrophic, acidophilic bacterium Thiobacillus ferrooxidans has been examined. Two rRNA operons were found in this microorganism by means of genomic hybridization studies. Recombinant plasmids, pTR‐3 and pTR‐1 that carry a portion of 16/23 S rDNA from one operon and the 5′‐flanking region of the second operon, respectively, were identified and characterized.
Some evidence of the enzymatic conversion of bovine suppressor phosphoseryl‐tRNA to selenocysteyl‐tRNA
Authors: Mizutani Takaharu
Published: July 2002

In order to clarify the mechanisms of selenocysteine incorporation into glutathione peroxidase, some evidence to show the in vitro conversion of phosphoseryl‐tRNA to selenocysteyl‐tRNA is reported. [3H]Phosphoseryl‐tRNA was incubated in a reaction mixture composed of SeO2, glutathione and NADPH in the presence of selenium‐transferase partially purified. Analyses of amino acids on the product tRNA showed that a part (4%) of [3H]phosphoseryl‐tRNA was changed to [3H]selenocysteyl‐tRNA. The conversion from seryl‐tRNAsu or major seryl‐tRNAIGA was not found. Selenium‐transferase was essential for the conversion. [3H]Selenocysteine, liberated from the tRNA, was modified with iodoacetic acid. The product was confirmed to be carboxymethyl‐selenocysteine by two‐dimensional TLC. Selenocysteyl‐tRNAsu should be used to synthesize glutathione peroxidase by co‐translational mechanisms.
Component X of mammalian pyruvate dehydrogenase complex: Structural and functional relationship to the lipoate acetyltransferase (E2) component
Authors: Neagle James , De Marcucci Olga , Dunbar Bryan , Lindsay J.Gordon
Published: July 2002

The lipoate acetyltransferase (E2, M
r 70 000) and protein X (M
r 51 000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N‐terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C‐labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly‐arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N‐terminus.
Finnish hereditary amyloidosis
Authors: Maury C.P.J. , Alii K. , Baumann M.
Published: July 2002

Amyloid fibrils were isolated from the kidney of a patient with Finnish hereditary amyloidosis. After solubilization of the fibrils in guanidine‐HCl, fractionation by gel filtration, and purification by reverse‐phase high‐performance liquid chrornatography, a homogeneous amyloid protein with an apparent M
r of 9000 was obtained. The protein was subjected to enzymatic digestion by trypsin and endoproteinase Lys‐C. The amino acid sequences were determined for 6 of the released peptides and they were all found to be identical to the reported, deduced primary structure of human plasma gelsoline in the region of amino acids 235–269. The results show that the amyloid fibril protein in Finnish hereditary amyloidosis represents a new type of amyloid protein that shows amino acid sequence homology with gelsoline, an actin‐modulating protein.
The 12 kDa protein of potato virus M displays properties of a nucleic acid‐binding regulatory protein
Authors: Gramstat Andrea , Courtpozanis Angelos , Rohde Wolfgang
Published: July 2002

The 3' terminal 1.4 kb segment of potato virus M (PVM) genomic RNA was cloned and sequenced. This part of the viral genome encodes the capsid protein CP as well as a 12 kDa protein of as yet unknown function. Both proteins were expressed in bacteria and their nucleic acid‐binding properties studied. The 12 kDa protein (prl2), but not the capsid protein bound single‐ and double‐stranded nucleic acids. This property of prl2 in conjunction with a zinc finger motif located adjacent to a basic region of the 12 kDa protein suggests that it may act as a regulatory factor during virus replication.
Kinetic study of esterification by immobilized lipase in n‐hexane
Authors: Chulalaksananukul W. , Condoret J.S. , Delorme P. , Willemot R.M.
Published: July 2002

The kinetic of the esterification of oleic acid by ethanol catalyzed by immobilized lipase of Mucor miehei in n‐hexane as a solvent has been completely studied. The kinetics of the reaction are suggested to agree with a Ping‐Pong Bi Bi mechanism in which only inhibition by excess of ethanol has been identified. Values of all apparent kinetic parameters were computed. No evidence of any significant external diffusional limitation which could account for these values has been detected. Optimization of water content through distribution ratio of water between solvent and support was examined.
Functional significance of sequences following the TATA box of an immunoglobulin promoter studied by random mutagenesis
Authors: Lieber Andre , Teppke Manfred , Herrmann Gottfried , Strauss Michael
Published: July 2002

We have investigated the importance of sequences downstream to the TATA box or an immunoglobulin promoter by transfection and in vitro transcription assays. A sequence from −11 to +10 with respect to the transcriptional start site was synthsised by a procedure allowing for random misincorporation of nucleotides. The pool of mutant oligonucleotides was cloned into the respective position of a vector carrying a fusion of a synthetic immunoglobulin heavy chain promoter with the human growth hormone gene. From 200 clones sequenced, 115 were mutants with at least one nucleotide exchange in every position. Whereas most mutations are of minor functional importance, changes at or near the transcriptional start site reduce the promoter activity considerably.
Effects of heat shock on the level of trehalose and glycogen, and on the induction of thermotolerance in Neurospora crassa
Authors: Neves Maria José , Jorge Joâo A. , François Jean M. , Terenzi Héctor F.
Published: July 2002

Neurospora crassa conidiospore germlings exposed to a heat shock (30–45°C) rapidly accumulated trehalose and degraded glycogen, even in the presence of cycloheximide. This phenomenon was also rapidly reversible upon return of the cells at 30°C. Trehalose accumulation at 45°C demanded an exogenous source of carbon and either glucose or glycerol fulfilled such requirement. Experiments with the cyclic AMP‐deficient cr‐1 mutant suggested that the effects of temperature shifts on trehalose level were independent of cAMP metabolism. Cells exposed at 45°C under conditions permissive for trehalose accumulation (i.e. in the presence of an assimilable carbon source) also acquired thermotolerance.
A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis
Authors: Ippoliti Rodolfo , Lendaro Eugenio , Bellelli Andrea , Brunori Maurizio
Published: July 2002

Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs). The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin‐biotin detection. The main complex (mol. wt. ≈ 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E. coli, a resistant species. This observation supports the hypotesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a ‘receptor’ site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.
Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthesis enzyme
Authors: Chamovitz Daniel , Misawa Norihiko , Sandmann Gerhard , Hirschberg Joseph
Published: July 2002

The first committed step in the biosynthetic pathway of carotenoids in plants and algae is the conversion of geranylgeranyl pyrophosphate (GGPP) to prephytoene pyrophosphate (PPPP), which is converted to phytoene. We have cloned the gene pys that encodes the enzyme phytoene synthase in the cyanobacterium Synechococcus PCC7942. The co‐expression of pys in cells of Escherichia coli together with the gene crtE from Erwinia uredovora, which encodes geranylgeranyl pyrophosphate synthase, resulted in accumulation of phytoene. This result indicates that phytoene synthase is a single polypeptide enzyme that catalyzes the 2‐step reaction from GGPP to phytoene. The deduced amino acid sequence of pys is highly conserved with that of pTOM5, a tomato cDNA that is differentially expressed during fruit ripening. These findings suggest that pTOM5 encodes phytoene synthase in tomato.
‘boxA’‐like sequence between the 16 S/23 S spacer in rRNA operon of mycoplasmas
Authors: Harasawa R. , Uemori T. , Asada K. , Kato I. , Shiragami N.
Published: July 2002

We have found that a boxA‐like sequence is conserved in the 16 S and 23 S rRNA intergenic spacer regions of mycoplasmas, and that it always locates on loop regions of the hypothetical secondary stem‐loop structures. A nucleotide sequence similar to the ‘−10’ box of prokaryotic promoters was identified at upstream sites of the boxA‐like sequence in the 16 S/23 S spacer regions. These structures may represent an internal promoter between the 16 S and 23 S rRNA genes in mycoplasmas.
Characterization of a multi‐copy gene for a major stage‐specific cysteine proteinase of Leishmania mexicana
Authors: Souza Augustine E. , Waugh Sheila , Coombs Graham H. , Mottram Jeremy C.
Published: July 2002

Imcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucel and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C‐terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi‐copy and tandemly arranged. Imcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage‐specific expression of LmCPb enzyme activity.
The nitrogenase MoFe protein
Authors: Gerloff Dietlind L. , Jenny Thomas F. , Knecht Lukas J. , Gonnet Gaston H. , Benner Steven A.
Published: July 2002

Surface residues, interior residues, and parsing residues, together with a secondary structure derived from these, are predicted for the MoFe nitrogenase protein in advance of a crystal structure of the protein, scheduled shortly to appear in Nature. By publishing this prediction, we test our method for predicting the conformation of proteins from patterns in the divergent evolution of homologous protein sequences in a way that places the method ‘at risk’.
Mos is degraded by the 26S proteasome in a ubiquitin‐dependent fashion
Authors: Ishida Naruhiro , Tanaka Keiji , Tamura Tomohiro , Nishizawa Mayumi , Okazaki Kenji , Sagata Noriyuki , Ichihara Akira
Published: July 2002

Mos, the c‐mos proto‐oncogene product, is a key regulator of cell cycle progression. Recently, rapid turnover of Mos in an early stage of meiotic maturation of Xenopus oocytes was found to be mediated by the ubiquitin pathway, but the protease responsible for its breakdown was not identified. In the present study, we found that 35S‐labeled Mos synthesized in an in vitro transcription/translation system was degraded ATP‐ and time‐dependently by the 26S proteasome, but not by the 20S proteasome, in the presence of a ubiquitin‐ligation system. The 26S proteasome did not degrade a mutant Mos in which Ser3 was replaced by Asp3 that is metabolically stable in oocytes, indicating a similarity in the proteolytic events in vivo to those observed in vitro in the present work. This is the first demonstration that the proteasome catalyzes the ATP‐dependent degradation of a naturally occurring, short‐lived oncoprotein by the ubiquitin pathway. This finding suggests that the proteasome may regulate the intracellular stability of various oncoproteins.
cDNA deduced procionin
Authors: Monstein Hans-Jürg , Thorup Jan Ulrik , Folkesson Ronnie , Johnsen Anders H. , Rehfeld Jens F.
Published: July 2002

Using an improved 3' RACE (PCR) amplification system containing oligonucleotide primer with an inosine at ambiguous codon positions and inverse PCR to amplify the 5' ends, we have isolated and characterized cDNA clones which encode cionin, a protochordean homologue of the mammalian hormones, cholecystokinin (CCK) and gastrin. The full‐length cloned cDNA of 510 bp encoded a 128 amino acid preprocionin. Reverse transcription‐PCR and subsequent cDNA cloning revealed that cionin mRNA is expressed in both the neuronal ganglion and the gut of the protochordate Ciona intestinalis. The primary structure of procionin resembles that of proCCK more than that of progastrin. Sequence‐specific immunochemical analysis showed that the cionin gene is expressed also at peptide level in both the gut and the neural ganglion. The neuronal processing of procionin is, however, more complete both with respect to carboxyamidation and tyrosine O‐sulfation. Hence, the tissue‐specific expression of the cionin gene in Ciona intestinalis resembles that of the CCK gene in mammals.
8‐(3‐Chlorostyryl)caffeine (CSC) is a selective A2‐adenosine antagonist in vitro and in vivo
Authors: Jacobson Kenneth A. , Nikodijević Olga , Padgett William L. , Gallo-Rodriguez Carola , Maillard Michel , Daly John W.
Published: July 2002

An adenosine antagonist, 8‐(3‐chlorostyryl)caffeine (CSC), was shown previously to be 520‐fold selective for A2a‐adenosine receptors in radioligand binding assays in the rat brain. In reversing agonist effects on adenylate cyclase, CSC was 22‐fold selective for A2a receptors in rat pheochromocytoma cells (K
b 60 nM) vs. A1 receptors in rat adipocytes (K
b 1.3 μM). Administered i.p. in NIH mice at a dose of 1 mg/kg, CSC shifted the curve for locomotor depression elicited by the A2a‐selective agonist APEC to the right (ED50 value for APEC shifted from 20 μg/kg i.p. to 190 μg/kg). CSC had no effect on locomotor depression elicited by an ED50 dose of the A1‐selective agonist CHA. CSC alone at a dose of 5 mg/kg stimulated locomotor activity by 22% over control values. Coadministration of CSC and the A1‐selective antagonist CPX, both at non‐stimulatory doses, increased activity by 37% (P < 0.001) over CSC alone, suggesting a behavioral synergism of A1‐ and A2‐antagonist effects in the CNS.
The binding site of the transcriptional activator VirG from Agrobacterium comprises both conserved and specific nonconserved sequences
Authors: Roitsch T. , Jin S. , Nester E.W.
Published: July 2002

Virulence genes of Agrobacterium tumefaciens are transcriptionally activated in response to phenolic compounds and certain sugars. The transcriptional activator VirG specifically binds to fragments containing the conserved vir box sequence present in the promoter region of all vir genes. This study shows that both the vir box as well as specific nonconserved sequences downstream of the vir box are required for VirG binding and transcriptional activation. Insertion of the identified VirG binding site into the lac promoter resulted in transcriptional activation of this heterologous promoter in response to the plant phenolic signal molecule acetosyringone.
Precise expression of the cAMP receptor gene, CAR1, during transition from growth to differentiation in Dictyostelium discoideum
Authors: Abe Fumiyoshi , Maeda Yasuo
Published: July 2002

The gene expressions associated with the switch‐over from cell proliferation of Dictyostelium discoideum to differentiation have been analyzed using temperature shift and differential plaque hybridization methods. Quit 1 was cloned as a specifically expressed gene when cells were starved just before the PS point (putative shift point from growth to differentiation). The coding region of the Quit 1 gene is identical to that of the cAMP receptor 1 CAR1 gene, which is essential for development. Quit1 mRNA was specifically expressed just after starvation of cells at around the PS point, thus indicating the importance of CAR1 for cellular differentiation as well as the actual existence of the PS point.
Epidermal growth factor influences the neurotrophic/differentiating action of nerve growth factor
Authors: Buravsky Vladimir A. , Gronskaya Raisa I. , Shpak Galina A. , Lustchitskaya Nina I. , Kalunov Vladimir N.
Published: July 2002

Exposure of naive PC12 cells, sympathetic neurons from rat superior cervical ganglia, and brain‐derived septal neurons to epidermal and nerve growth factors simultaneously resulted in some alteration of cellular events induced by nerve growth factor alone. A more pronounced decline of catecholamine content, no additional change in acetylcholinesterase activity, and additive stimulation of RNA and protein syntheses were found in PC12 cells. Earlier elevation of the enzyme activity was observed in sympathetic but not in septal neurons. Epidermal growth factor appeared to support independently the same level of acetylcholinesterase activity in septal neurons as revealed for nerve growth factor during the first week and cell survival throughout 2 weeks of observation. The data obtained indicate that epidermal growth factor augments temporarily some effects of nerve growth factor, thus supporting the idea of an important role of mitogenic growth factors in neural development as complementary and/or substitutive regulators of nerve cell differentiation and survival.
Maturation of cerebellar granule cells is delayed in cultures derived from ethanol‐treated chick embryos: Survival and proliferation studies
Authors: Srivastava Nisha , Vernadakis Antonia
Published: July 2002

Previous studies from this laboratory have shown that ethanol administration to chick embryos during embryonic days 1–3, a critical period of neuroembryogenesis, differentially affects primordial CNS structures. In this study, chick embryos were treated in ovo with ethanol (10 mg/50 μl/day) at E1 to E3. At 14 days of embryonic age cerebellar (E14CE) granule cell cultures were prepared from both control and ethanol‐treated embryos. Growth patterns were evaluated morphologically and the neuronal nature of these cultures was evaluated immunocytochemically. E14CE granule cell cultures exhibited neurofilament immunoreactivity demonstrating the neuronal‐nature of these cultures. In addition E14CE granule cultures contained numerous glutamateric neurons as assessed by positive glutamate immunoreactivity and also some GABAergic neurons as assessed by positive GABA immunoreactivity. Cultures derived from both control and ethanol‐treated embryos were labeled with 3H‐thymidine and assessed for effects on survival and proliferation in culture. Cultures derived from ethanol‐treated embryos showed a higher rate of proliferation and survival during the first 3 days in culture as compared to those derived from controls. However, after 3 days in culture, survival was lower in the cultures from ethanol‐treated embryos as compared to those derived from controls. We interpret these findings to mean that (a) ethanol arrested cerebellar granule cell development at an immature state; (b) immature neurons have a higher survival capacity than differentiated neurons; and (c) ethanol accelerates normal neuronal cell death as previously reported.
Effect of a spinal cord photolesion injury on catalase
Authors: Sampath D. , Holets V. , Perez‐Polo J.R.
Published: July 2002

Ischemic injury to the spinal cord results in cell and tissue damage. Oxygen free radicals have been implicated in post‐ischemic cell injury and death while free radical scavengers like superoxide dismutase and catalase are associated with an amelioration of ischemic injury. Measurement of catalase enzyme activity or protein in ischemic tissue presents mechanical problems due to extensive tissue destruction. Therefore, we looked at the effects of a photochemical lesion (which reproduces ischemic injury) on the levels of catalase mRNA in the spinal cord tissues of rodents under various experimental conditions. A significant depletion in the levels of catalase mRNA was observed in the spinal cord tissues of rats that received a severe lesion and were sacrificed 6 days post‐lesion, while levels of catalase mRNA in the spinal cord tissues of similarly lesioned rats sacrificed 14 days post‐lesion showed a return to control values.
Reinnervation of denervated iris by transplanted sympathetic ganglia: Effect of neuronal age
Authors: Hiebert Jean , Smith Peter G.
Published: July 2002

Neuronal outgrowth in vivo is aggressive postnatally, but is diminished with increasing age. This may be attributable to intrinsic features of the neuron or its interaction with other components of the developing organism. The purpose of this study was to determine if there is an age‐dependent reduction in the intrinsic ability of sympathetic neurons to initiate fiber outgrowth. Superior cervical ganglia from donor rats aged 3–4, 11–12, 27–28 and 45–46 days were removed and transplanted to the anterior chamber of the sympathectomized eye of host rats 85–89 days of age. Ganglia with host irides were removed at 3, 6 and 10 days post‐transplant and whole mounts were analysed using catecholamine histofluorescence for maximum sympathetic fiber density, length and initial rate of outgrowth. Fluorescent fibers were present in host irides of donors of all ages and at all post‐transplant times. However, maximum fiber density was less for the 3–4 day‐old donor ganglia (e.g. 43–71% of 11–46‐day‐old donor ganglia at 600 μm, 10 days post‐transplant). Maximum fiber length was also less in the youngest group (e.g. 35–49% of 11–46‐day‐old donor ganglia, 10 days post‐transplant). Further, the initial rate of outgrowth was decreased for the 3–4‐day‐old donor ganglia (128±46 μm/day for the 3–4‐day‐old ganglia vs 253±48 μm/day for the 11–12‐day‐old ganglia, 307±35 μm/day for the 27–28‐day‐old ganglia and 260±22 μm/day for the 45–46‐day‐old ganglia). We conclude that the capacity for neuronal outgrowth is limited in the early postnatal period, but rapidly attains the mature level by the second postnatal week. Therefore, diminished outgrowth in mature rats does not appear to be due to a decrease in the intrinsic ability of sympathetic nerves to initiate fiber outgrowth, but is attributable to other factors.
Overexpression of T‐type calcium channels in HEK‐293 cells increases intracellular calcium without affecting cellular proliferation
Authors: Chemin Jean , Monteil Arnaud , Briquaire Christelle , Richard Sylvain , Perez-Reyes Edward , Nargeot Joël , Lory Philippe
Published: July 2002

Increased expression of low voltage‐activated, T‐type Ca2+ channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T‐type α1 subunits, α1G, α1H and α1I, now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human α1G and α1H subunits in human embryonic kidney (HEK‐293) cells, we describe here that, although T‐type channels mediate increases in intracellular Ca2+ concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T‐type Ca2+ channels are not sufficient to modulate cellular proliferation of HEK‐293 cells.
Characterization of wheat DP, a heterodimerization partner of the plant E2F transcription factor which stimulates E2F–DNA binding
Authors: Ramirez-Parra Elena , Gutierrez Crisanto
Published: July 2002

Recent studies suggest that the G1/S transition in plants depends on the activity of E2F transcription factors. In animal cells, E2Fs interact with DP proteins, whose identification in plants has been elusive, so far. Here we show that although an E2F‐containing DNA‐binding activity can be detected in plant cell extracts, purified E2F protein binds poorly to DNA. In a yeast two‐hybrid screening, using wheat E2F as a bait, we have isolated a cDNA clone encoding a wheat DP (TmDP) protein. TmDP is expressed ubiquitously and exhibits a domain organization similar to animal DPs. Contrary to the specificity observed for the plant RBR/E2F interaction, human and plant E2F and DP proteins can interact in a heterologous manner. Purified TmDP protein stimulates E2F–DNA complex formation.
Mapping of protein‐protein interactions between c‐myb and its coactivator CBP by a new phage display technique
Authors: Kiewitz Andreas , Wolfes Heiner
Published: July 2002

We have developed a phage display technique for the mapping of protein‐protein interaction sites and characterized the interaction between the c‐myb proto‐oncogene product and its co‐activator CBP. Arbitrary DNA segments of the c‐myb gene were cloned into a modified phagemid which allowed for expression in all possible reading frames. The mini‐library encompassing all functional domains of the protein was propagated as phages and screened with different bait proteins. Alignment of the sequences revealed that the amino acids 317–342 of Myb interact with the CBP protein. Furthermore, an intramolecular interaction of the N‐terminal Myb DNA binding domain with the C‐terminus (amino acids 541–567) could be detected.
YDL142c encodes cardiolipin synthase (Cls1p) and is non‐essential for aerobic growth of Saccharomyces cerevisiae
Authors: Tuller Gabriele , Hrastnik Claudia , Achleitner Georg , Schiefthaler Ursula , Klein Franz , Daum Günther
Published: July 2002

The unassigned open reading frame YDL142c was identified to code for cardiolipin synthase, Cls1p. A cls1 deletion strain is viable on glucose, galactose, ethanol, glycerol and lactate containing media, although the growth rate on non‐fermentable carbon sources is decreased. Mitochondria of the cls1 mutant are devoid of cardiolipin but accumulate the cardiolipin precursor phosphatidylglycerol when grown on non‐fermentable carbon sources. Specific activity of phosphatidylglycerolphosphate synthase in cls1 is reduced to 30–75% of the wild‐type level. Amounts of mitochondrial cytochromes and activity of cytochrome c oxidase, however, are not affected in the cls1 deletion strain. Collectively, these data indicate that cardiolipin is not essential for aerobic growth of Saccharomyces cerevisiae.
Self‐oligomerization of NACP, the precursor protein of the non‐amyloid β/A4 protein (Aβ) component of Alzheimer's disease amyloid, observed in the presence of a C‐terminal Aβ fragment (residues 25–35)
Authors: Paik Seung R , Lee Ju-Hyun , Kim Do-Hyung , Chang Chung-Soon , Kim Young-Sik
Published: July 2002

NACP, the precursor protein of the non‐amyloid β/A4 protein (Aβ) component of Alzheimer's disease (AD) amyloid, also known as α‐synuclein, was suggested to seed amyloid plaque formation in AD by stimulating Aβ aggregation. We have demonstrated that NACP experienced self‐oligomerization only in the presence of a modified Aβ fragment (Aβ25–35) by using dicyclohexylcarbodiimide. This NACP oligomerization, appearing as a discrete ladder on a Tricine SDS‐PAGE, was not observed with other Aβ peptides such as the reverse peptide Aβ35–25 and Aβ1–40, indicating this process was specific not only for the C‐terminal peptide sequence of the Aβ but also for its orientation. It might be, therefore, suggested that the NACP self‐oligomers formed only in the presence of a N‐terminally truncated Aβ peptide could act as a nucleation center for plaque formation during AD development.
Activation of protein phosphatase 2A by cAMP‐dependent protein kinase‐catalyzed phosphorylation of the 74‐kDa B″ (δ) regulatory subunit in vitro and identification of the phosphorylation sites
Authors: Usui Hirofumi , Inoue Rintaro , Tanabe Osamu , Nishito Yasumasa , Shimizu Masahiro , Hayashi Hideyuki , Kagamiyama Hiroyuki , Takeda Masao
Published: July 2002

Human erythrocyte protein phosphatase 2A, which comprises a 34‐kDa catalytic C subunit, a 63‐kDa regulatory A subunit and a 74‐kDa regulatory B″ (δ) subunit, was phosphorylated at serine residues of B″ in vitro by cAMP‐dependent protein kinase (A‐kinase). In the presence and absence of 0.5 μM okadaic acid (OA), A‐kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B″, respectively. The K
m value of A‐kinase for CAB″ was 0.17±0.01 μM in the presence of OA. The major in vitro phosphorylation sites of B″ were identified as Ser‐60, ‐75 and ‐573 in the presence of OA, and Ser‐75 and ‐573 in the absence of OA. Phosphorylation of B″ did not dissociate B″ from CA, and stimulated the molecular activity of CAB″ toward phosphorylated H1 and H2B histones, 3.8‐ and 1.4‐fold, respectively, but not toward phosphorylase a.
Demonstration of a tandem pair of complement protein modules in GABAB receptor 1a
Authors: Hawrot Edward , Xiao Yuanyuan , Shi Qing-luo , Norman David , Kirkitadze Marina , Barlow Paul N.
Published: July 2002

We have subcloned and expressed the N‐terminal portion of the recently sequenced metabotropic GABA receptor, GABABR1a. This region of the receptor contains a complement protein‐like amino acid sequence. The purified 140‐residue recombinant protein fragment was soluble and stable. Mass spectrometry indicated formation of four disulfide bonds, as expected if two complement protein modules (CPs, also known as SCRs, Sushi domains) are formed. The circular dichroism spectrum was unusual and characteristic of CPs. Differential scanning calorimetry demonstrated a melting point (64°C), and total enthalpy commensurate with two fully folded domains. We thus conclude that the 1a subtype of the GABAB receptor, but not the 1b subtype, contains a pair of CPs and we present a three‐dimensional model of this region.
Identification of a novel actin binding site within the Dp71 dystrophin isoform
Authors: Howard Perry L. , Klamut Henry J. , Ray Peter N.
Published: July 2002

The Dp71 dystrophin isoform has recently been shown to localize to actin filament bundles in early myogenesis. We have identified an actin binding motif within Dp71 that is not found in other dystrophin isoforms. Actin overlay assays and transfection of COS‐7 cells with fusion proteins of wild type and mutated Flag epitope‐tagged Dp71 demonstrate that this motif is necessary and sufficient to direct localization of Dp71 to actin stress fibers. Furthermore, this localization is independent of alternative splicing which alters the C‐terminus of the protein. The identification of an actin binding site suggests Dp71 may function to anchor membrane receptors to the cytoskeleton.
Tyrosine phosphorylation and translocation of LAT in platelets
Authors: Sarkar Sibaji
Published: July 2002

Platelet aggregation is accompanied by the tyrosine phosphorylation of several proteins including syk. However, some of these proteins are not identified. Recent studies showed that LAT is a syk substrate and is tyrosine phosphorylated during T cell stimulation. In this study, we demonstrated that LAT is present in platelets and is tyrosine phosphorylated in response to ADP‐ and thrombin‐stimulated aggregation. Moreover, LAT, like syk and β3, translocates to the cytoskeleton during the late stage of thrombin‐stimulated irreversible aggregation and not during ADP‐stimulated reversible aggregation.
Prorenin processing by cathepsin B in vitro and in transfected cells
Authors: Jutras Isabelle , Reudelhuber Timothy L.
Published: July 2002

Renin, which catalyzes the initial proteolytic cleavage reaction in the production of angiotensins, is first synthesized as a zymogen, prorenin, and requires the proteolytic removal of an amino‐terminal prosegment for activation in vivo. The lysosomal hydrolase cathepsin B has been proposed as a prorenin processing enzyme based on reports of its co‐localization with renin in the secretory granules of certain tissues and its ability to activate prorenin in vitro. In the current study, scanning mutagenesis was used to identify the amino acids which determine the site selectivity of prorenin cleavage by human cathepsin B in vitro. Co‐expression assays in AtT‐20 cells were also used to test for the ability of cathepsin B to cleave human prorenin within cells. Our results suggest that a basic lysine residue at the −2 position from the cleavage site is required for cathepsin B cleavage of prorenin in vitro and that the structure of prorenin itself may account for the selection of the proper cleavage site. In addition, although cathepsin B appears to be correctly sorted to lysosomes, the enzyme exhibits prorenin processing activity in transfected AtT‐20 cells, raising the question of the cellular localization in which the processing event occurs.
Differential regulation of uncoupling protein‐1, ‐2 and ‐3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold‐exposed rats
Authors: Denjean Frédérique , Lachuer Joël , Géloën Alain , Cohen-Adad Frédérique , Moulin Colette , Barré Hervé , Duchamp Claude
Published: July 2002

The control of uncoupling protein‐1, ‐2 and ‐3 (UCP‐1, UCP‐2, UCP‐3) mRNA levels by sympathetic innervation in rats was investigated by specific and sensitive RT‐PCR assays. In rats reared at thermoneutrality (25°C), unilateral surgical sympathetic denervation of interscapular brown adipose tissue (BAT) markedly reduced the UCP‐1 mRNA level (−38%) as compared with the contralateral innervated BAT pad, but was without significant effect on UCP‐2 and ‐3 mRNA levels. Cold exposure (7 days, 4°C) markedly increased UCP‐1 (+180%), UCP‐2 (+115%) and UCP‐3 (+195%) mRNA levels in interscapular BAT. Unilateral sympathetic denervation prevented the cold‐induced rise in BAT UCP‐1 and UCP‐2 mRNAs, but not that in BAT UCP‐3 mRNA. Results were confirmed by Northern blot analysis. These data indicate a differential endocrine control of UCP‐1, UCP‐2 and UCP‐3 gene expression in rat BAT both at thermoneutrality and during prolonged cold exposure.
Arginine‐aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro
Authors: Litovchick Alexander , Evdokimov Artem G , Lapidot Aviva
Published: July 2002

HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat‐transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of l‐arginine with aminoglycosides. Using a gel‐shift assay and affinity chromatography on an l‐arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with K
d values in the range of 20–400 nM, which is comparable to the K
d of native Tat bound to the transactivation responsive element (10–12 nM). Confocal microscopy studies demonstrated that fluorescein‐labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design.
Pseudoazurin mediates periplasmic electron flow in a mutant strain of Paracoccus denitrificans lacking cytochrome c550
Authors: Koutný Marek , Kučera Igor , Tesařı́k Radek , Turánek Jaroslav , Van Spanning Rob J.M.
Published: July 2002

A periplasmic protein able to transfer electrons from cytoplasmic membrane to the periplasmic nitrite reductase (cytochrome cd1
) has been purified from the anoxically grown cytochrome c550
mutant strain Pd2121 and shown to be pseudoazurin by several independent criteria (molecular mass, copper content, visible spectrum, N‐terminal amino acid sequence). Under our assay conditions, the half‐saturation of electron transport occurred at about 10 μM pseudoazurin; the reaction was retarded by increasing ionic strength.
Recognizing the Pleckstrin homology domain fold in mammalian phospholipase D using hidden Markov models
Authors: Holbrook Pamela G , Geetha V , Beaven Michael A , Munson Peter J
Published: July 2002

Phospholipase D was first described in plant tissue but has recently been shown to occur in mammalian cells where it is activated by cell surface receptors. Its mode of activation by receptors in unclear. Biochemical studies suggest that it may occur downstream of other effector proteins and that small GTP‐dependent regulatory proteins may be involved. The sequence in a non‐designated region of mammalian phospholipase D1 and 2 shows similarity to a structural domain that is present in signalling proteins that are regulated by protein kinases or heterotrimeric G‐proteins. Mammalian phospholipase D has structural similarities with other lipid signalling phospholipases and thus may be regulated by receptors in an analogous fashion.
Two novel delta‐endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus
Authors: Wojciechowska J.A , Lewitin E , Revina L.P , Zalunin I.A , Chestukhina G.G
Published: July 2002

Genes cry26Aa1 and cry28Aa1 were cloned from Bacillus thuringiensis ssp. finitimus strain B‐1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aa1 gene product included seven residues determined to be an N‐terminal part of a chymotrypsin‐treated delta‐endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore‐associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aa1 in both types of crystal bodies is synthesized under the control of one and the same genomic locus.
Localization of calponin binding sites in the structure of 90 kDa heat shock protein (Hsp90)
Authors: Bogatcheva Natalia V , Ma YuShu , Urosev Dunja , Gusev Nikolai B
Published: July 2002

The structure of rabbit liver Hsp90 was reevaluated by limited trypsinolysis, N‐terminal sequencing and determination of the site that is phosphorylated by casein kinase II. Limited proteolysis results in formation of four groups of large peptides withMrin the range of 26–41 kDa. Peptides withMr39–41 kDa were represented by large N‐terminal and central peptides starting at residue 283 of the α‐isoform of Hsp90. All sites phosphorylated by casein kinase II were located in the large 39–41 kDa peptides. Peptides withMr26–27 kDa were represented by short N‐terminal and central peptides starting at Glu‐400 of the α‐isoform of Hsp90. The data of affinity chromatography and light scattering indicate that smooth muscle calponin interacts with Hsp90. The calponin binding sites are located in the large (37–41 kDa) N‐terminal and in a short (26–27 kDa) central peptide starting at Glu‐400 of the α‐isoform of Hsp90. Phosphorylation by casein kinase II up to 2 mol of phosphate per mol of Hsp90 does not affect interaction of Hsp90 with calponin.
Thermodynamics of nucleotide binding to NBS‐I of the Bacillus subtilis preprotein translocase subunit SecA
Authors: den Blaauwen Tanneke , van der Wolk Jeroen P.W. , van der Does Chris , van Wely Karel H.M. , Driessen Arnold J.M.
Published: July 2002

SecA is the dissociatable nucleotide and preprotein binding subunit of the bacterial translocase. The thermodynamics of nucleotide binding to soluble SecA at nucleotide binding site I were determined by isothermal titration calorimetry. Binding of ADP and non‐hydrolyzable ATPγS is enthalpy‐driven (ΔH
0 of −14.44 and −5.56 kcal/mol, respectively), but is accompanied by opposite entropic contributions (ΔS
0 of −18.25 and 9.55 cal/mol/K, respectively). ADP binding results in a large change in the heat capacity of SecA (ΔC
p=−780 cal/mol/K). It is suggested that ADP binding promotes the interaction between the two thermodynamically discernible domains of SecA which is accompanied by a shielding of hydrophobic surface from solvent.
OTC and AUL1, two convergent and overlapping genes in the nuclear genome of Arabidopsis thaliana
Authors: Quesada Vı́ctor , Ponce Marı́a Rosa , Micol José Luis
Published: July 2002

In contrast to bacterial, fungal and vertebrate ornithine transcarbamylases (OTCs; EC 2.1.3.3), very little is known about the enzyme in plants. We report here the isolation of a T‐DNA‐tagged mutant displaying sensitivity to ornithine, whose characterization has allowed for the identification of several complementary and genomic DNA clones encoding the OTC and auxilin‐like 1 (AUL1) proteins of the crucifer Arabidopsis thaliana. Transcript mapping revealed that at least 22 bp within the OTC‐AUL1 intercoding region are transcribed from both strands, which makes this one of the rarely described cases of convergent and overlapping transcription units in the nuclear genome of a multicellular eukaryote. Transcription of the OTC gene was shown to be ubiquitous in aerial organs of adult plants, whereas that of AUL1 was obscured by the existence of a putative second copy of the gene. The OTC‐AUL1 locus maps at the bottom of chromosome 1.
Long‐term microglial and astroglial activation in the hippocampus of trimethyltin‐intoxicated rat: stimulation of NGF and TrkA immunoreactivities in astroglia but not in microglia
Authors: Koczyk Danuta , Oderfeld‐Nowak Barbara
Published: July 2002

In the present study we investigated the microglial and astroglial response after trimethyltin (TMT) exposure over a prolonged period of time. Male Wistar rats were given a single dose of TMT (8 mg/kg, i.p.) and survived 4, 7, 21, 60 and 180 days after the administration of the toxin. Histochemistry (Griffonia simplicifolia lectinstaining) and immunocytochemistry for GFAP were applied to identify micro‐ and astroglial cells, respectively. To assess the trophic response of glial cells (NGF and TrkA expression), single or double staining experiments were performed. In addition, the biochemical evaluation of GFAP and NGF were carried out at chosen timepoints using immunoblotting technique and ELISA, respectively. The main findings of our study were as follows. (1) A protracted activation of microglia (at least up to 2 months posttreatment). (2) A long‐lasting expression of GFAP immunoreactivity (at least up to 6 months posttreatment) and a steady increase in GFAP content (at least up to 2 months posttreatment). (3) The appearance of enormously enlarged, round‐shape astrocytes exclusively localized to CA1 and observed 2 months posttreatment. (4) The stimulation of NGF and TrkA expression in reactive astrocytes. (5) The strongest activation of micro‐ and astroglia coincided with the most prominent neurodegeneration in the hippocampus, i.e., in CA4/CA3c and CA1. It is tempting to assume that the activation of glial cells in the hippocampal areas particularly vulnerable to TMT may affect neuronal fate after neurotoxic insult.
Increased allergic reactivity of atopic type in mood disorders and schizophrenia
Authors: Dabkowska M , Rybakowski JK
Published: July 2002

The allergic reactivity of atopic type was investigated in 54 patients with mood disorders in the course of depressive episode and in 39 patients with schizophrenia. The assessment was done by three methods: intradermal tests, determination of IgE concentration and determination of specific immunoglobulin E (IgE) antibodies. The hypersensitivity to three or more allergens on intradermal tests was found in 40% of depressive and in 27% of schizophrenic patients. The concentration of IgE > 100 IU/mL was found in 45% of patients with depression and in 46% of patients with schizophrenia, more frequently in subjects with hyperactivity of hypothalamic-pituitary-adrenal axis, and in depression, in patients with higher intensity of symptoms. The positive results of the Phadiatop test which detects specific antibodies for inhalatory antigens, were found in 46% of depressive and in 40% of schizophrenic patients The indices of atopy for all three tests were higher in males than in females in both mood disorders and in schizophrenia. However, these indices were similar for unipolar and bipolar depressed patients as well as for patients with paranoid or residual schizophrenia.
Onset of action under antidepressant treatment
Authors: Stassen H.H. , Angst J , Delini-Stula A
Published: July 2002

The issues of timing in antidepressant treatment are of great theoretical and practical relevance, even more so since recent meta-analyses yielded no evidence for a specific mode of action of antidepressants, which, according to the theory of delayed onset of action, is expected to emerge after 2 weeks of therapy. To address the issues of timing on a methodologically sound basis, future trials should adapt a ‘longitudinal’ rather than ‘cross-sectional’ design, standardized with respect to a washout period, baseline and first 2 week assessments. With this in mind, special attention should be paid to parameters which potentially enable the identification of placebo responders, true drug responders and patients at risk of non-improvement. Results and methods of the Zurich meta-analyses may serve as a starting point for further steps in this direction.
Near‐Infrared II Plasmonic Au@Au–Ag Dot‐in‐Cubic Nanoframes for In Vivo Surface‐Enhanced Raman Spectroscopic Detection and Photoacoustic Imaging
Authors: Shan Beibei , Li Linhu , Zhao Yawen , Wang Haitao , Li Ming
Published: May 2021

Near‐infrared II (NIR‐II) plasmonic noble‐metallic nanostructures are highly desirable for in vivo photodiagnostics because of their multiple functionalities and deep‐tissue optical imaging capability. Herein, a novel class of Au@Au–Ag dot‐in‐cubic nanoframes (DCFs) comprising a solid Au core and a Au–Ag alloy shell with a wall hole on each side face is reported, which exhibit remarkable plasmonic properties spectrally tunable beyond 1400 nm while maintaining a small size. The synthesis of Au@Au–Ag DCFs is principally based on the facet‐selective etching of Ag atoms and co‐deposition of Au and Ag atoms on the edges and corners of Au@Ag dot‐in‐cubes. The geometry (e.g., edge length and wall hole size) can be controllably tailored by simply varying the silver nitrate and chloroauric acid concentrations. Superior in vitro and in vivo NIR‐II photoacoustic imaging performances of Au@Au–Ag DCFs are demonstrated. Additionally, remarkable NIR‐II surface‐enhanced Raman spectroscopy (SERS) activity of Au@Au–Ag DCFs is evidenced with the maximum NIR‐II SERS enhancement occurring at a plasmon resonance wavelength blue‐shifting from the incident laser wavelength and an excellent effective detection depth. Finally, the potential of the developed Au@Au–Ag DCFs is highlighted for in vivo NIR‐II SERS detection of microtumors in animals.