bluuebunny/crossref_metadata_2025 huggingface dataset:
Mammalian genomic sequences can substitute for the SV40 AT stretch in sustaining replication of the SV40 origin of replication
Authors: Galli Ivo , Iguchi-Ariga Sanae M.M. , Ariga Hiroyoshi
Published: July 2002

The core of the SV40 origin of replication (ori) contains a stretch of adenine (A) and thymine (T) residues. This region is very conserved among the papova viruses, and is known to be extremely sensitive to mutations. So far, mutations have been found to drastically reduce, and in most cases abolish, replication. The AT stretch has been shown to be the target for several host cellular proteins that belong to the replication machinery. We reasoned that, in this light, there might exist cellular DNA sequences that can substitute for the SV40 AT stretch. To study this possibility, we digested mammalian genomic DNA and inserted the fragments instead of the SV40 AT stretch in a plasmid carrying the SV40 ori core. The resulting pool was analyzed by a ‘replication trap’ in CosI cells. We present evidence that there are indeed several mammalian sequences that can substitute for the SV40 AT stretch. All of them are rich in adenines and thymines but, surprisingly, these sequences differ from the wild‐type SV40 AT stretch to such extent that at first sight they would seem unlikely to replicate. This is all the more impressive f we consider that another AT‐rich sequence from the yeast TRP1 gene, which also carries a similar variation, cannot substitute for the SV40 AT stretch.
Two structural genes are encoding malate synthase isoenzymes in Saccharomycescerevisiae
Authors: Fernandez Ernestina , Fernandez Maria , Rodicio Rosaura
Published: July 2002

We report on the isolation of a gene encoding yeast malate synthase. A yeast genomic library was screened using a probe homologous to the yeast enzyme obtained by the polymerase chain reaction. The nucleotide sequence of the cloned gene was determined. Computer analysis showed that the isolated gene is identical to the one previously described as DAL7, which is involved in allantoin metabolism [Mol. Cell. Biol. 9 (1989) 3231‐3243]. Enzymatic activities of multicopy transformants, Southern analysis and disruption mutants predict the existence of two genes encoding malate synthases that are differentially regulated at the transcriptional level. Malate synthase; Glyoxylate pathway; Allantoin metabolism; Gene disruption.
Translation and fates of the gag protein of 1731, a Drosophila melanogaster retrotransposon
Authors: Kim Myeong-Hee , Coulondre Christine , Champion Serge , Lacoste Jérôme , Best-Belpomme Martin , Maisonhaute Claude
Published: July 2002

An entire copy of 1731, a Drosophila melanogaster retrotransposon, was tagged by fusing in frame its putative gag gene with the reporter LacZ sequence. The high transfection efficiency of Drosophila virilis cells added to the absence of 1731 in their genome allowed, by combining histochemical staining and immunological detections, the demonstration of the translation of the 1731 gag gene. The gag protein is gathered in virus‐like particles. Its occurrence in nuclei is consistent with a nuclear localization signal. The expression of the sense construction was inhibited by cotransfections with its antisense homologue.
Mature carnitine palmitoyltransferase I retains the N‐terminus of the nascent protein in rat liver
Authors: Kolodziej Marian P. , Zammit Victor A.
Published: July 2002

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full‐length cDNA sequence of the protein. One peptide was found to correspond to the extreme N‐terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N‐terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N‐terminus of the mature protein allows a reappraisal of the role of the two main, N‐terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.
Kinetics of CO binding to H+‐motive oxidases of the caa3‐type from Bacillus FTU and of the o‐type from Escherichia coli
Authors: Muntyan M.S. , Bloch D.A. , Ustiyan V.S. , Drachev L.A.
Published: July 2002

The kinetics of CO rebinding with isolated Bacillus FTU
caa
3‐type oxidase and with solubilized Escherichia coli membranes (GO103 strain) containing the o‐type oxidase as the main O2reducing enzyme were studied under reducing conditions by laser flash photolysis of the CO‐oxidase complexes. The spectra of the optical absorbance changes upon photolysis were characteristic of CO‐caa
3 and CO‐o‐oxidase complexes in Bac. FTU and E.Coli, respectively. Small quantities of d‐type oxidase in E.Coli GO103 membranes were detected. The kinetics of CO reassociation with reduced caa
3 and o‐type oxidases were monophasic with τ 25–30 ms in both cases.
Simultaneous solubilization of high‐affinity receptors for VIP and glucagon and of a low‐affinity binding protein for VIP, shown to be identical to calmodulin
Authors: Andersson Mats , Carlquist Mats , Maletti Maura , Marie Jean-Claude
Published: July 2002

Anion‐exchange chromatography of solubilized pig liver cell membranes on DEAE‐Sepharose gave a fraction with high affinity binding proteins for VIP and glucagon distinct from each other. Scatchard analysis indicated the presence of one binding site for VIP (K
d 1.5 ± 0.6 nM and B
max 1.3 ± 0.4 pmol/mg). The order of potency for VIP‐related peptides to inhibit [125I]VIP binding was: VIP > peptide histidine isoleucine amide (PHI) > rat growth hormone releasing factor (rGRF) > secretin. GTP‐γ‐S inhibited [125I]vip binding and reduced the affinity of VIP binding sites to 6.5 nM. In the same isolated fraction, [125I]glucagon binding was displaced by glucagon preferentially to oxyntomodulin, and GTP did not affect this [125I]glucagon binding. Scatchard analysis indicated the presence of one binding site for glucagon (K
d 0.08 ± 0.03 nM and B
max 0.31 ± 0.01 pmol/lg). A low‐affinity VIP binding protein (IC50 0.7 μM) was detected in a fraction eluting later and exhibited a peptide specificity: rGRF > VIP > VIP(10–28) > secretin > PHI. This rGRF‐preferring protein (18 kDa) was purified and had a partial amino‐acid sequence identical to that of calmodulin. Its [125I]VIP binding was competitively inhibited by VIP and calmidazolium in a manner similar to that for pig brain calmodulin. Thus we have co‐solubilized VIP and glucagon high affinity receptors from pig liver cell membranes and separated them from VIP‐binding calmodulin.
F0 and F1 parts of ATP synthases from Clostridium thermoautotrophicum and Escherichia coli are not functionally compatible
Authors: Das Amaresh , Ljungdahl Lars G.
Published: July 2002

F1‐stripped membrane vesicles from Clostridium thermoautotrophicum and Escherichia coli were reconstituted with F1‐ATPases from both bacteria. Reconstituted FiFo‐ATPase complexes were catalytically active, i.e. capable of hydrolyzing ATP. Homologous‐type ATPase complexes having F0 and F1 parts of ATP synthases from the same origin were DCCD sensitive and supported ATP‐driven enhancement ofanilinonaphthalene sulfonate (ANS) fluorescence. Hybrid‐type ATPase complexes having F0 and F1 parts of ATP synthases from different origins were neither DCCD sensitive nor did they support ATP‐driven enhancement of ANS fluorescence. Analyzing these results it has been demonstrated that the F0 and F1 parts of ATP synthases of these two bacteria are not functionally compatible.
Subunit‐specific block of cloned NMDA receptors by argiotoxin636
Authors: Raditsch Martin , Ruppersberg J.Peter , Kuner Thomas , Günther Willy , Schoepfer Ralf , Seeburg Peter H. , Jahn Werner , Witzemann Veit
Published: July 2002

Cloned NMDA receptor channels of the NR1‐NR2A, NR1‐NR2B and NR1‐NR2C type show differences in argiotoxin636 block. Mutations of an asparagine residue located at a homologous position in the TM2 region of all NMDA receptor subunits, which corresponds to the Q/R site of the AMPA receptors, alters the argiotoxin636‐induced block. The results suggest that the toxin interacts at this amino acid position with the putative pore forming TM2 region of the NMDA receptor subunits. Sequence differences in the TM2 segment of NR2A and NR2C subunits are not responsible for the subtype‐specific sensitivity to argiotoxin636 as revealed by site‐directed mutagenesis.
Expression of non‐hepatic‐type S‐adenosylmethionine synthetase isozyme in rat hepatomas induced by 3′‐methyl‐4‐dimethylaminoazobenzene
Authors: Hirokawa Saburo , Kobayashi Yuki , Sugiyama Toshihiro , Terashima Hiromichi , Wada Kenji , Tsukada Kinji
Published: July 2002

It is known that a high incidence of hepatocellular carcinoma in rat liver can be induced by such azo dye carcinogens as 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB). Mammalian S‐adenosylmethionine (AdoMet) synthetase exists as two isozymes, non‐hepatic‐type and liver‐type enzymes, which are the products of two different genes. We have examined the expression of two AdoMet synthetase isozyme proteins and mRNAs in rat hepatomas induced by 3′‐Me‐DAB. The levels of non‐hepatic‐type enzyme protein and mRNA are clearly induced by 3′‐Me‐DAB feeding. On the other hand, the levels of liver‐type enzyme protein and mRNA are nearly the same or slightly decreased during hepatocarcinogenesis. These results indicate that the expression of the non‐hepatic‐type isozyme gene is obviously influenced with the progression of carcinogenesis and that the non‐hepatic‐type isozyme is useful as a oncodevelopmental marker in the liver.
Molecular cloning of cDNA for the 29 kDa proteinase participating in decomposition of the larval fat body during metamorphosis of Sarcophaga peregrina (flesh fly)
Authors: Takahashi Noboru , Kurata Shoichiro , Natori Shunji
Published: July 2002

A cDNA clone for the 29 kDa proteinase participating in tissue disintegration during metamorphosis of Sarcophaga was isolated. This proteinase, named Sarcophaga cathepsin B, consisted of 256 amino acid residues, and contained three putative N‐glycosylation sites. By comparison with other cathepsins B, its unique substrate specificity was partly explained by Ala at position 248.
Downregulation of phospho‐tyrosine phosphatases in a macrophage tumor
Authors: Sridhar T.S. , Swarup G. , Khar Ashok
Published: July 2002

We provide evidence for the downregulation of phospho‐tyrosine phosphatases (PTPases) in malignancy. AK‐5, a rat macrophage tumor, shows the downregulation of the transcripts of two non‐receptor‐type PTPases, PTP‐1 and PTP‐S. Though downregulated fourfold, the genomic organization of PTP‐S is unaltered. There is no gross alteration of the PTPase activity in AK‐5 as compared to macrophages. Immunoblot analysis reveals no significant change in the total phospho‐tyrosine levels in AK‐5, but there is a qualitative difference in the pattern between AK‐5 and macrophages. Our results lend credence to the conjecture that PTPases also might be involved in malignancy.
Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions
Authors: Petersen Lars C. , Schiødt Jakob , Christensen Ulla
Published: July 2002

Des(1–38) factor VIIa and des(1–44) factor VIIa were obtained by limited proteolysis. The binding of tissue factor to these factor VIIa‐derivatives was assessed from its stimulation of the proteolytic activity on chromogenic obgopeptide substrates. Compared to native factor VIIa (K
TF = 0.6 ± 0.1 nM), Tissue factor binds to des(1–38) factor VIIa with a lower, but still significant affinity (K
TF = 4.8 ± 0.3 nM). The activity of des(1–44) factor VIIa was only slightly stimulated by TF (K
TF ∼ 200 nM). Binding of TF depends critically on the presence of Ca2+ ions. Ca2+ ions stimulated the activity of factor VIIa/TF with an apparent K
Ca = 0.16 ± 0.02 mM. Factor VIIa in the absence of tissue factor was stimulated by Ca2+ with an apparent K
Ca = 0.05 ± 0.01 mM, and similar K
Ca values were obtained for the truncated derivatives of factor VIIa. Measurements of Ca2+‐induced changes in intrinsic protein fluorescence suggest a conformational change. The Ca2+ ion concentration at which this change occurred was higher for des(1–44) factor VIIa (apparent K
Ca = 0.14 mM) than for des(1–38)‐ and native factor VIIa (apparent K
Ca = 0.04 mM). The Tb3+ ion luminescence technique was used to further investigate the Ca2+ binding sites. Tb3+ ions bound with a lower affinity to des(1–44) factor VIIa than to des(1–38)‐and native factor VIIa. The observed drastic decrease in affinity for tissue factor as a result of truncation of the ‘hydrophobic stack’ residues 39–44, suggest that this region of factor VIIa provides a structural determinant that together with other regions participates in tissue factor binding.
Plant proteins that bind to the 3′‐terminal sequences of the negative‐strand RNA of three diverse positive‐strand RNA plant viruses
Authors: Hayes R.J. , Pereira V.C.A. , Buck K.W.
Published: July 2002

The replication of positive‐strand RNA plant viruses, which involves both virus‐encoded and plant‐encoded proteins, takes place in two stages: synthesis of a negative‐strand RNA using the genomic positive‐strand RNA as a template and synthesis of progeny positive‐strand RNA using the negative‐strand RNA as a template. Using gel mobility shift and photochemical crosslinking assays, we have identified three proteins of M
r 32K, 50K and 100K in extracts of tobacco and spinach leaves that bind to the 3′‐terminal sequences of the negative‐strand RNA of three diverse positive‐strand RNA plant viruses. The 32K protein was purified to near homogeneity by chromatography on columns of Macro‐prep high Q, heparin‐sepharose, single‐stranded DNA cellulose and poly(U)‐sepharose. No binding of any of the three proteins to the 3′‐termini of the positive‐strand RNA or the 5′‐termini of the positive‐strand RNA or negative‐strand RNA of any of the three viruses, or to the 3′‐termini of the mRNAs of two chloroplast genes, psbA or petD, could be detected. We propose that 3′‐terminal negative‐strand RNA binding proteins, which may be widespread in the plant kingdom, could be utilised by at least three different positive‐strand RNA plant viruses for the initiation of positive‐strand RNA synthesis.
A leucine motif in the amino acid sequence of subunit 9 of the mitochondrial ATPase, and other hydrophobic membrane proteins, that is highly conserved by editing
Authors: Konstantinov Yuri M. , Møller Ian M.
Published: July 2002

Subunit 9 of the mitochondrial ATPase, but also other hydrophobic mitochondrially encoded proteins, contains a high frequency of the leucine motif, ‐Leu‐X9‐Leu‐, which is highly conserved through RNA editing. The leucine motif may provide specific recognition sites between membrane‐spanning domains of the F0‐ATPase and between other hydrophobic subunits during the assembly of multienzyme complexes in the inner mitochondrial membrane.
Photophobic responses and phototaxis in Chlamydomonas are triggered by a single rhodopsin photoreceptor
Authors: Kröger Petra , Hegemann Peter
Published: July 2002

The rhodopsin nature of the photoreceptor for the behavioural light responses in Chlamydomonas has originally been revealed by action spectroscopy. Meanwhile most physiological experiments and the identification of all‐trans‐retinal in cell extracts favour that this chlamyrhodopsin contains an all‐trans‐type retinal chromophore with strong similarity to the light sensors SR I and SRII from Halobacteria. Reconstitution of retinal‐deficient cells with [3H]retinal identified a single retinal protein with a MW of 30,000. Chlamyrhodopsin triggers a photoreceptor current in the eyespot region resulting in direction changes or phototaxis. Furthermore, when the light stimulus oversteps a critical level, two flagellar currents appear, which are the basis for photophobic responses. The physiological, electrophysiological and biochemical experiments suggest that all behavioural responses are triggered by a single rhodopsin‐type receptor.
Identification of glutamine and lysine residues in Alzheimer amyloid βA4 peptide responsible for transglutaminase‐catalysed homopolymerization and cross‐linking to α2M receptor
Authors: Rasmussen Lone K. , Sørensen Esben S. , Petersen Torben E. , Gliemann Jørgen , Jensen Poul Henning
Published: July 2002

The β‐amyloid peptide (βA4), derived from a larger amyloid precursor protein, is the principal component of senile plaques in Alzheimer's disease. Here we report that the full‐length (1–40) synthetic βA4 peptide, containing one glutamine and two lysine residues, is able to form homopolymers in a transglutaminase‐mediated reaction. Moreover, transglutaminase catalysed the formation of heteropolymers in reactions of βA4 with α2M receptor, a constituent of amyloid plaques, and with extracellular matrix proteins. Incorporation of site‐specific probes followed by enzymatic digestion and sequencing of tracer‐containing fractions demonstrated that both Lys16, Lys28 and Gin15 in βA4 were susceptible to cross‐linking by transglutaminase.
Analysis of revertants from respiratory deficient mutants within the center N of cytochrome b in Saccharomyces cerevisiae
Authors: Coppée Jean-Yves , Tokutake Nobuya , Marc Denis , di Rago Jean-Paul , Miyoshi Hideto , Colson Anne-Marie
Published: July 2002

Four modified cytochrome b's carrying mononucleotide substitutions affecting center N residues were analysed. The mutant carrying a G33D change does not incorporate heme into the apocytochrome b and fails to grow on non‐fermentable carbon sources. Out of 85 genetically independent revertants derived from this mutant, 82 were true back‐mutants restoring the wild type sequence (D33G). The remaining three replaced the aspartic acid by an alanine (D33A) indicating that small size residues are best tolerated at this position which is consistent with the perfect conservation of the G33 during evolution. This glycine may be of crucial importance for helix packing around the hemes. The replacement of methionine at position 221 by lysine (M221K.) produced a non‐functional cytochrome b [(1993) J. Biol. Chem. 268,15626‐15632]. Non‐native revertants replacing the lysine 221 by glutamic acid (K221E) or glutamine (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b residues in 33 and in 221 seemed to contribute to the quinone reduction (Q
N
) site of the cytochrome bc
1 complex. Possible intramolecular interactions between the N‐tenninal region and the loop connecting helices IV and V of cytochrome b are proposed.
The expression of PDGF‐α but not PDGF‐β receptors is suppressed in Swiss/3T3 fibroblasts over‐expressing protein kinase C‐α
Authors: Fitzer-Attas Cheryl , Eldar Hagit , Eisenbach Lea , Livneh Etta
Published: July 2002

The generation and characterization of Swiss/3T3 cells which stably over‐express protein kinase C (PKC)‐α were previously described by us. In these cells over‐expression of PKC‐α reduced the expression of epidermal growth factor (EGF) receptor molecules [(1990) J. Biol. Chem. 265, 13290‐13296]. Here we show that the expression of PDGF‐α receptors, but not PDGF‐β receptors, was specifically decreased in these cells. Not only were the levels of PDGF‐α receptor mRNA transcript and protein significantly diminished in the PKC‐α over‐producing cells, but their ability to respond to short‐ and long‐term growth factor signals was appropriately compromised. This was reflected in a reduced tyrosine autophosphorylation signal in response to PDGF‐AA, as well as in decreased growth rates of PKC‐α over‐expressing cells when supplied with external PDGF‐AA. A similar decrease in PDGF‐α receptors was also demonstrated in parental Swiss/3T3 cells treated with phorbol esters. Our studies imply that PKC‐α is involved in a cellular mechanism suppressing the expression of PDGF‐α receptors in Swiss/3T3 cells. Hence, activation of PKC‐α or alterations in its cellular levels may affect, in turn, the expression of a specific set of cell surface receptors and their responses to external growth factors.
Functional expression of Cre recombinase in sub‐regions of mouse CNS and retina
Authors: Eriksson Björn , Bergqvist Ingela , Eriksson Maria , Holmberg Dan
Published: July 2002

We describe a strategy for generating CNS and retina sub‐region‐specific mutations using the Cre/loxP system. Transgenic mice expressing Cre recombinase under the control of the c‐kit promoter were established. Functional Cre expression was predominantly found to be restricted to the CA1, CA2 and CA3 regions of the hippocampus, the anterior region of the dentate gyrus, and to the ganglion cell layer of the retina.
Fatty acid 9‐ and 13‐hydroperoxide lyases from cucumber1
Authors: Matsui Kenji , Ujita Chika , Fujimoto Sho-hei , Wilkinson Jack , Hiatt Bill , Knauf Vic , Kajiwara Tadahiko , Feussner Ivo
Published: July 2002

Fatty acid hydroperoxide lyase (HPL) is a novel P‐450 enzyme that cleaves fatty acid hydroperoxides to form short‐chain aldehydes and oxo‐acids. In cucumber seedlings, the activities of both fatty acid 9HPL and 13HPL could be detected. High 9HPL activity was especially evident in hypocotyls. Using a polymerase chain reaction‐based cloning strategy, we isolated two HPL‐related cDNAs from cucumber hypocotyls. One of them, C17, had a frameshift and it was apparently expressed from a pseudogene. After repairing the frameshift, the cDNA was successfully expressed in Escherichia coli as an active HPL with specificity for 13‐hydroperoxides. The other clone, C15, showed higher sequence similarity to allene oxide synthase (AOS). This cDNA was also expressed in E. coli, and the recombinant enzyme was shown to act both on 9‐ and 13‐hydroperoxides, with a preference for the former. By extensive product analyses, it was determined that the recombinant C15 enzyme has only HPL activity and no AOS activity, in spite of its higher sequence similarity to AOS.
Analysis of dipeptidyl peptidase IV gene regulation in transgenic mice: DNA elements sufficient for promoter activity in the kidney, but not the intestine, reside on the proximal portion of the gene 5′‐flanking region
Authors: Gum James R. , Erickson Roger H. , Hicks James W. , Rius Jose L. , Kim Young S.
Published: July 2002

The dipeptidyl peptidase IV (DPPIV) gene encodes a brush border membrane exopeptidase that is expressed in a tissue‐restricted fashion. To examine the regulation of DPPIV transcription in various tissues in vivo, we examined the expression of DPPIV 5′‐flanking region (promoter)–human growth hormone reporter constructs in transgenic mice. These mice exhibited cell‐type specific reporter expression in kidney. Surprisingly, however, only very low to non‐detectable levels of reporter were found in small intestine. These results indicate that DNA elements sufficient for DPPIV expression in kidney, but not intestine, reside in the 5′‐flanking region of the gene.
Recombinant C1b domain of PKCδ triggers meiotic maturation upon microinjection in Xenopus laevis oocytes
Authors: Aroca Pilar , Santos Eugenio , Kazanietz Marcelo G
Published: July 2002

The C1 domains are 50 amino acid sequences present in protein kinase C (PKC) isozymes that are responsible for binding of phorbol esters and the lipid second messenger diacylglycerol (DAG). We found that bacterially expressed C1b domain of PKCδ induces germinal vesicle breakdown (GVBD) when microinjected into Xenopus laevis oocytes. Injection of the C1b domain of PKCδ significantly enhanced insulin‐ but not progesterone‐induced maturation. Interestingly, the PKCδ C1b domain markedly synergized with normal Ras protein to induce oocyte maturation when both proteins were co‐injected in oocytes. Our results demonstrate that the purified C1b domain of PKCδ is sufficient to promote meiotic maturation of X. laevis oocytes probably through activation of components of the insulin/Ras signaling pathway.
Cloning of Plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor 1,4‐bis{3‐[N‐(cyclohexyl methyl)amino]propyl}piperazine 1
Authors: Florenta Isabelle , Mouray Elisabeth , Dali Ali Fouad , Drobecq Hervé , Girault Sophie , Schrével Joseph , Sergheraert Christian , Grellier Philippe
Published: July 2002

A series of 10 1,4‐bis(3‐aminopropyl)piperazine compounds was found to display antiplasmodial activity with 50% growth inhibition between 30 and 250 nM, on three Plasmodium falciparum strains differently sensitive to chloroquine. By affinity chromatography using one of these compounds, a 52‐kDa protein was isolated from P. falciparum, microsequenced and cloned. It corresponded to a single copy gene encoding a 453 amino acid protein displaying the typical features of protein disulfide isomerases, a thiol metabolizing enzyme belonging to the thiol: disulfide oxidoreductase superfamily, which was not previously described in malarial species.
Arginine kinase of the flagellate protozoa Trypanosoma cruzi
Authors: Alonso Guillermo D , Pereira Claudio A , Remedi Marı́a S , Paveto M.Cristina , Cochella Luisa , Ivaldi M.Soledad , Gerez de Burgos Nelia M , Torres Héctor N , Flawiá Mirtha M
Published: July 2002

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas’ disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme‐specific activity and enzyme protein was observed. Arginine kinase‐specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post‐transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.
Identification of a C‐terminal binding site for G‐protein βγ‐subunits in phosducin‐like protein
Authors: Schröder Stefan , Blüml Klaus , Dees Christian , Lohse Martin J
Published: July 2002

Phosducin‐like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G‐protein βγ‐subunit (Gβγ)‐mediated signaling, with an affinity about 5‐fold lower than that of phosducin. The Gβγ binding site of phosducin has been suggested to be contained in its N‐terminus. A region corresponding to this N‐terminus is lacking in PhLP, suggesting that PhLP must utilize a different mode of Gβγ binding. To map the Gβγ binding site in PhLP, a series of deletion mutants were constructed, expressed in E. coli as glutathione S‐transferase (GST) fusion proteins, and the purified fusion proteins were examined for their ability to attenuate Go GTPase activity. Progressive N‐terminal truncations of PhLP caused only minor reductions in potency, whereas the complementary N‐terminal PhLP fragments turned out to be inactive. We further identified a short C‐terminal segment comprising residues 168 to 195 that inhibited Go GTPase activity similar in efficacy and potency to full‐length PhLP. This C‐terminal fragment was also capable of antagonizing a second Gβγ‐mediated function, the enhancement of rhodopsin phosphorylation by the β‐adrenergic receptor kinase. Taken together, these data indicate that PhLP interacts with Gβγ via a short C‐terminal binding site which is distinct from that identified previously in phosducin.
Nitric oxide mediates NMDA‐evoked [3H]GABA release from chick retina cells
Authors: Ientile Riccardo , Pedale Santa , Picciurro Vincenzo , Macaione Vincenzo , Fabiano Caterina , Macaione Salvatore
Published: July 2002

The stimulation of NMDA receptor increased [3H]GABA release from preloaded cultured retina cells. This effect appears to be mediated by NO production, since addition of L‐NA reduces NMDA‐evoked [3H]GABA release. Spermine/NO complex, an NO donor, mimics the effect produced by NMDA. The addition of zaprinast, a phosphodiesterase inhibitor, as well as 8‐Br‐cGMP enhances the NMDA‐evoked [3H]GABA release. These results agree with the existence in chick retina cells of NO/cGMP pathways and support a role for NO in NMDA‐evoked events. The activation of this receptor complex through maturative stages of the retina together with the NO‐mediated increase in GABA release may account for NMDA differentiative effect in culturing retina cells.
Promoter‐independent regulation of cell‐specific dopamine receptor expression
Authors: Knapp Mark , Wong Albert H.C , Schoots Oscar , Guan H.C , Van Tol Hubert H.M
Published: July 2002

Here we describe the construction of recombinant adenoviruses expressing dopamine D2 and D4 receptors, and their ability to mediate high levels of heterologous expression in a variety of cell types in vitro and in vivo for at least 7 days post infection. These experiments demonstrated that maximum receptor expression is achieved generally within 24 h and remains constant thereafter. Maximum expression levels were highly variable between cell lines and dependent on infection efficiency and promoter strength. Correction for these two variables revealed differences in relative expression levels between cell lines varying by two orders of magnitude. Our results indicate that in addition to gene transcription, post‐transcriptional mechanisms play a dominant role in determining dopamine receptor levels in this system.
ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells
Authors: Murata-Hori Maki , Suizu Futoshi , Iwasaki Takahiro , Kikuchi Asako , Hosoya Hiroshi
Published: July 2002

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper‐interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642–1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin‐activated MgATPase activity of myosin II. HeLa ZIPK is the first non‐muscle MLCK that phosphorylates MRLC at two sites.
Induction of cyclic AMP and cyclic GMP 3′:5′‐cyclic nucleotide phosphodiesterase activities in neuroblastoma lines under differentiating conditions
Authors: Giorgi Mauro , Giordano Daniela , Caniglia Cristiana , Biagioni Stefano , Augusti‐Tocco Gabriella
Published: July 2002

It is now widely accepted that cyclic nucleotide phosphodiesterases (PDEs) play fundamental roles in signal transduction pathways; they show a remarkable molecular complexity, different tissue distribution and complex regulatory mechanisms. Here we report PDE isoforms expression in two dibutyryl cyclic AMP differentiated murine cell lines: the hybrid neuroblastoma‐glioma 108CC15 and the parental neuroblastoma N18TG2. They differ for the ability to establish functional synapses, a feature present only in the former. Ionic exchange chromatography elution profiles of N18TG2 and 108CC15 undifferentiated cell extracts show two main peaks of activity. The first one hydrolyzes cyclic GMP and is specifically inhibited by Zaprinast, thus representing a member of the PDE5 family. The second peak hydrolyzes cyclic AMP and is significantly inhibited by rolipram, as all the PDE4 family members. The induction of differentiation by dibutyryl cyclic AMP in both clonal lines results in an increase of PDE activities only after 3 hr of treatment, suggesting that protein neosynthesis is involved. Interestingly in both clones, besides the increase in cyclic AMP hydrolyzing specific activity (3.1 folds in 108CC15 and 2.5 folds in N18TG2), we also observed an increase in cyclic GMP hydrolyzing activity (1.7 folds in 108CC15 and 4.3 folds in N18TG2). While the induction of PDE4, previously reported also in other cellular systems, could be considered as a feedback response to the higher cyclic AMP levels, this is not true for the isoform that hydrolyzes cyclic GMP. These data suggest that the induction of PDE isoforms in neuroblastoma cells could be related to the activation of neuronal differentiative pathway.
Ca2+ mobilization and capacitative Ca2+ entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells
Authors: Sugioka Miho , Zhou Wen‐Liang , Hofmann Hans‐Dieter , Yamashita Masayuki
Published: July 2002

Release of Ca2+ from intracellular Ca2+ stores (Ca2+ mobilization) and capacitative Ca2+ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca2+ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5‐di‐tert‐butylhydroquinone (DBHQ), an inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and of SK&F 96365, an inhibitor of capacitative Ca2+ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [3H]‐thymidine incorporation in a dose‐dependent manner without affecting cell viability or morphology. The inhibition of [3H]‐thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC50: ∼4 μM) as the blockade of capacitative Ca2+ entry in the E3 retinal organ culture. At a concentration of 5 μM SK&F 96365, DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca2+ stores also inhibited [3H]‐thymidine incorporation with IC50 values of 20–30 μM in the different cultures. Our results suggest the involvement of Ca2+ mobilization and capacitative Ca2+ entry in the regulation of DNA synthesis in the developing neural retina.
Automatic segmentation of rectal tumor on diffusion‐weighted images by deep learning with U‐Net
Authors: Zhu Hai‐Tao , Zhang Xiao‐Yan , Shi Yan‐Jie , Li Xiao‐Ting , Sun Ying‐Shi
Published: August 2021

Purpose

Manual delineation of a rectal tumor on a volumetric image is time‐consuming and subjective. Deep learning has been used to segment rectal tumors automatically on T2‐weighted images, but automatic segmentation on diffusion‐weighted imaging is challenged by noise, artifact, and low resolution. In this study, a volumetric U‐shaped neural network (U‐Net) is proposed to automatically segment rectal tumors on diffusion‐weighted images.

Methods

Three hundred patients of locally advanced rectal cancer were enrolled in this study and divided into a training group, a validation group, and a test group. The region of rectal tumor was delineated on the diffusion‐weighted images by experienced radiologists as the ground truth. A U‐Net was designed with a volumetric input of the diffusion‐weighted images and an output of segmentation with the same size. A semi‐automatic segmentation method was used for comparison by manually choosing a threshold of gray level and automatically selecting the largest connected region. Dice similarity coefficient (DSC) was calculated to evaluate the methods.

Results

On the test group, deep learning method (DSC = 0.675 ± 0.144, median DSC is 0.702, maximum DSC is 0.893, and minimum DSC is 0.297) showed higher segmentation accuracy than the semi‐automatic method (DSC = 0.614 ± 0.225, median DSC is 0.685, maximum DSC is 0.869, and minimum DSC is 0.047). Paired t‐test shows significant difference (T = 2.160, p = 0.035) in DSC between the deep learning method and the semi‐automatic method in the test group.

Conclusion

Volumetric U‐Net can automatically segment rectal tumor region on DWI images of locally advanced rectal cancer.
Strong, Ultrastretchable Hydrogel‐Based Multilayered Soft Actuator Composites Enhancing Biologically Inspired Pumping Systems
Authors: Banerjee Hritwick , Sivaperuman Kalairaj Manivannan , Ren Hongliang , Jusufi Ardian
Published: August 2021

Diverse solutions for active fluid movement are known in nature and in human‐made devices. However, commercial peristaltic pumps are mostly rigid, noncompliant, and tough to integrate into biocompatible materials. This work aims to approximate actuator‐like behavior concerning nonhemolytic pumping action and higher energy density to develop biorobotic physical models and biomedical assistive devices with life‐like motion profiles. To achieve this, dielectric elastomers (DEs) offer themselves. DE connected via very high bonding (VHB) tape's pumping performance is tested and compared to a novel configuration. Comparative analysis of the VHB‐based DE pump vis‐a‐vis the novel design solution involving composite layering of hydrogel and electroactive polymer (HEAP) with interfacial toughness of ≈1522 ± 188 J m−2 exhibits increases in pressure change of up to 68 mmHg at measured flow rates of 16.8 mL s−1 with low viscoelastic losses (% at biaxial prestretch of 3 × 3, 10% stretch rate, and 20 cycles postoperation). The HEAP‐sandwiched layer embracing hydrogel‐based ionotronics presents 2,205% ultimate strain and sustains compressive stress of 632 kPa. This pilot thus demonstrates the advantages of greater incorporation of hydrogel‐based biocompatible polymers in conjunction with soft active materials and proposes performance characterization for cardiovascular trials and related biofluid pumping applications.
Penetration Resistance of Cast Metal−Ceramic Composite Lattice Structures
Authors: Umanzor Manuel E. , Batra Romesh C. , Williams Christopher B. , Druschitz Alan P.
Published: August 2021

A challenging issue in armor mechanics is to optimize the impact resistance of a target per unit areal mass. Herein, the penetration resistance of an A356 alloy–ceramic lattice structure with ceramic tiles encapsulated in the metal matrix is experimentally and computationally studied to achieve this objective. A hybrid additive manufacturing/metal‐casting technique is used to fabricate the structure. The performance is experimentally evaluated by impacting the tiles at normal incidence with 0.30‐cal armor‐piercing projectiles and 7.62 M80 rounds traveling at high speed. X‐ray imaging and electron microscopy techniques are used to ascertain the quality of the castings and the damage caused to the target by the projectiles. The cast material is tested following ASTM standard E8/E8M to determine the yield stress and hardening coefficients in the Johnson–Cook model. Large deformations of the components are analyzed using the finite element software LS‐DYNA. The penetrator's computed residual velocities differ from their test values by less than 4% for the 0.30‐cal projectiles and 19% for the ball rounds. The effects of several design variables (e.g., tile thickness and location, among others) are numerically scrutinized. The proposed design is ≈20% lighter than the solid metal target to achieve the same residual velocity of the penetrator.
Fabrication of Carbon‐Bonded Alumina Filters by Additive‐Manufactured, Water‐Soluble Polyvinyl Alcohol Filter Templates and Alginate‐Based Slips
Authors: Bock-Seefeld Benjamin , Wetzig Tony , Hubálková Jana , Schmidt Gert , Abendroth Martin , Aneziris Christos G.
Published: August 2021

Since solid, nonmetallic inclusions considerably impair the mechanical properties of cast steel products, the removal of inclusions within micrometer range constitutes a major challenge. A new approach for the production of carbon‐bonded alumina filters is investigated. Water‐soluble polyvinyl alcohol (PVA) filter templates are fabricated with the aid of additive manufacturing and coated with alginate‐based slips. The coated filter templates are placed in an aqueous solution enriched with Ba2+ ions, triggering the gelation of the alginate‐based slip and the dissolution of the template. The hollow filters are subsequently coated twice with an alginate‐free slip and fired once at 800 °C. To evaluate the coating procedure, the filter samples are analyzed with regard to the filter structure and the mechanical properties after the coating steps. The results reveal that carbon‐bonded alumina filter with a sufficient dimensional filter stability can be produced without a thermal template removal. Thereby, a cold crushing strength of 0.13 MPa is achieved using additive‐manufactured PVA filter templates and an alginate‐based coating slip with a solid content of 55.6 wt% and an alginate content of 0.65 wt%.
Sputtering and Characterization of MAX‐Phase Forming Cr–Al–C and Ti–Al–C Coatings and Their Application on γ‐Based Titanium Aluminides
Authors: Laska Nadine , Bauer Peter-Philipp , Helle Oliver , Kreps Frederic
Published: August 2021

MAX‐phases are of increasing interest as coating material for high temperature applications due to their unique metallic as well as ceramic properties. Herein, the deposition of Cr2AlC and Ti3AlC2 or Ti2AlC MAX‐phase forming coatings by magnetron sputtering is demonstrated. Using pure elemental targets, the manufacturing with a coating thickness of above 7 μm is established. The MAX‐phase forming coatings are characterized by high‐temperature X‐ray diffraction (HT‐XRD) measurements and provide a good oxidation behavior due to the development of protective thermally grown oxide layers. The performance of the MAX‐phases is strongly depended on the substrate material and the accompanying interdiffusion processes. Therefore, the Ti–Al–C coating is favored for TiAl alloys due to the thermodynamic stability of the Ti2AlC MAX phase in particular in the presence of the γ‐TiAl phase. An excellent oxidation behavior is confirmed up to 300 h at 850 °C due to the development of an alumina layer above a homogenous Ti2AlC phase coating. The Cr2AlC MAX‐phase coating degrades after 100 h at 800 °C due to interdiffusion processes between coating and substrate and the accompanying development of carbides and nitride phases. Nevertheless, the oxidation resistance of the Cr–Al–C‐coated TiAl alloy is given by the formation of the Ti2AlC MAX‐phase.
Biomimetic Lamellar Chitosan Scaffold for Soft Gingival Tissue Regeneration
Authors: Feng Yanhuizhi , Gao Huai‐Ling , Wu Di , Weng Yu‐Teng , Wang Ze‐Yu , Yu Shu‐Hong , Wang Zuolin
Published: August 2021

Mucogingival surgery has become a common procedure for soft gingival tissue reparation in dental clinical practice, which mainly relies on autograft or commercial collagen membranes (CM). However, the autograft faces grand challenges in source availability and long‐term post‐surgery pain management, and the CM is restricted by its poor mechanical properties in an aqueous environment. Here, it is reported that a bio‐inspired lamellar chitosan scaffold (LCS) with long range ordered porous structure, manufactured through a bidirectional freezing method, can serve as a promising gingival tissue engineering material. The LCS not only exhibits excellent mechanical properties in the hydrated state but also accelerates vessel formation and soft tissue regeneration in vivo. Most interestingly, the LCS is found to be capable of inducing macrophage differentiation to M2 macrophages, which is thought to play an important role in tissue regeneration. These advantages combined with its easy and low‐cost preparation process make the LCS a promising candidate for dental clinical applications.
Biomimetic Glycopolypeptide Hydrogels with Tunable Adhesion and Microporous Structure for Fast Hemostasis and Highly Efficient Wound Healing
Authors: Teng Lin , Shao Zhengwei , Bai Qian , Zhang Xueliang , He Yu‐Shi , Lu Jiayu , Zou Derong , Feng Chuanliang , Dong Chang‐Ming
Published: August 2021

Despite clinical applications of the first‐generation tissue adhesives and hemostats, the correlation among microstructure and hemostasis of hydrogels with wound healing is less understood and it is elusive to design high‐performance hydrogels to meet worldwide growing demands in wound closure, hemostasis, and healing. Inspired by the microstructure of extracellular matrix and mussel‐mimetic chemistry, two kinds of coordinated and covalent glycopolypeptide hydrogels are fabricated, which present tunable tissue adhesion strength (14.6–83.9 kPa) and microporous structure (8–18 µm), and lower hemolysis <1.5%. Remarkably, the microporous size mainly controls the hemostasis, and those hydrogels with larger pores of 16–18 µm achieve the fastest hemostasis of ≈14 s and the lowest blood loss of ≈6% than fibrin glue and others. Moreover, both biocompatibility and hemostasis affect wound healing performance, as assessed by hemolysis, cytotoxicity, subcutaneous implantation, and hemostasis and healing assays. Importantly, the glycopolypeptide hydrogel‐treated rat‐skin defect model achieves full wound closure and regenerates thick dermis and epidermis with some hair follicles on day 14. Consequently, this work not only establishes a versatile method for constructing glycopolypeptide hydrogels with tunable adhesion and microporous structure, fast hemostasis, and superior healing functions, but also discloses a useful rationale for designing high‐performance hemostatic and healing hydrogels.
Coexistence of Negative and Positive Photoconductivity in Few‐Layer PtSe2 Field‐Effect Transistors
Authors: Grillo Alessandro , Faella Enver , Pelella Aniello , Giubileo Filippo , Ansari Lida , Gity Farzan , Hurley Paul K. , McEvoy Niall , Di Bartolomeo Antonio
Published: August 2021

Platinum diselenide (PtSe2) field‐effect transistors with ultrathin channel regions exhibit p‐type electrical conductivity that is sensitive to temperature and environmental pressure. Exposure to a supercontinuum white light source reveals that positive and negative photoconductivity coexists in the same device. The dominance of one type of photoconductivity over the other is controlled by environmental pressure. Indeed, positive photoconductivity observed in high vacuum converts to negative photoconductivity when the pressure is raised. Density functional theory calculations confirm that physisorbed oxygen molecules on the PtSe2 surface act as acceptors. The desorption of oxygen molecules from the surface, caused by light irradiation, leads to decreased carrier concentration in the channel conductivity. The understanding of the charge transfer occurring between the physisorbed oxygen molecules and the PtSe2 film provides an effective route for modulating the density of carriers and the optical properties of the material.
Laserjet Printed Micro/Nano Sensors and Microfluidic Systems: A Simple and Facile Digital Platform for Inexpensive, Flexible, and Low‐Volume Devices
Authors: Bamshad Arshya , Cho Hyoung Jin
Published: August 2021

A facile and digital do‐it‐yourself technique is proposed to fabricate inexpensive sensors on flexible substrates (paper, cloth, and plastic film). A set of office‐grade equipment (i.e., laserjet printer, thermal laminator, computer‐aided paper cutter), and commercially available supplies (i.e., baking wax paper, furniture restoration metal‐leaf) are used. Forming electrodes through traditional printing and defining a fluidic confinement region through crafting practice enable fabrication of a wide range of devices without requiring customized specialty instruments, costly infrastructure, and complicated fabrication steps, unlike previously introduced methods. Three different levels of experiments are designed to assess the comprehensiveness and responsiveness of the proposed method to the needs of existing research fields. The performances of the fabricated features at each level are evaluated to cover various application domains in environmental monitoring and biomedical diagnostics utilizing conductometric, colorimetric, biochemical, and chemoresistive detection principles. Devices with varying size of features, from nanometers to centimeters, are fabricated and characterized. This method provides an alternative route to decentralized production of low‐cost flexible sensors and other devices, with a minimal step, time, and facilities. The operation of such devices is simple and can be further empowered by smartphones for data analysis and transmission.