bluuebunny/crossref_metadata_2025 huggingface dataset:
Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids
Authors: Schoefs Benoı̂t , Rmiki Nour-Eddine , Rachadi Jaouad , Lemoine Yves
Published: July 2002

Astaxanthin accumulation by green microalgae is a natural phenomenon known as red snows and blood rains. The fact that astaxanthin synthesis requires oxygen, NADPH and Fe2+ led Cunningham and Gantt [Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 557–583] to propose that a cytochrome P450‐dependent enzyme might be involved in the transformation of β‐carotene to astaxanthin. In Haematococcus only esterified astaxanthin molecules accumulate, but it is not determined whether a fatty acid synthesis should occur simultaneously to allow pigment accumulation. The aim of this contribution was to answer these two questions using specific inhibitors of β‐carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine).
A model for Fis N‐terminus and Fis‐invertase recognition
Authors: Tzou Wen-Shyong , Hwang Ming-Jing
Published: July 2002

In this modeling, we show that the elusive Fis N‐terminus involved in invertase‐mediated site‐specific DNA inversion is, by all indications, a hinged flapping loop. The prediction is based on a combined sequence and secondary structure alignment against known structures of protein segments, as well as a tetrapeptide fragment observed crystallographically. Its validity is strongly supported by the ability to interpret consistently the available mutagenesis data pertaining to this region including, especially, a series of deletion mutants which until this work had been a puzzle in the search for structural explanations. A model for Fis‐invertase recognition is also proposed.
Preferential condensation of SAR‐DNA by histone H1 and its SPKK containing octapeptide repeat motif
Authors: Khadake Jyoti R , Rao M.R.S
Published: July 2002

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA·dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI‐SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3–4‐fold higher than that of the random DNA fragment. A 16‐mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C‐terminus of histone H1d, which has recently been shown to possess DNA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1‐SAR DNA interaction through SPKK containing peptide motifs and its competition by AT‐hook peptides present in the nonhistone chromosomal proteins like HMG‐I and HMG‐Y.
Protein tyrosine phosphatase activity modulation by endothelin‐1 in rabbit platelets
Authors: Catalán R.Edgardo , Gargiulo Laura , Martı́nez Ana M , Calcerrada M.Carmen , Liras Antonio
Published: July 2002

Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin‐1 stimulation of rabbit platelets resulted in a dose‐ and time‐dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70–100 and 100–200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose‐dependent inhibition of the endothelin‐stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6‐fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti‐protein tyrosine phosphatase‐1C from endothelin‐stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase‐1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin‐1 in rabbit platelets can be proposed through a cAMP‐dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.
The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells
Authors: Léon Catherine , Hechler Béatrice , Vial Catherine , Leray Claude , Cazenave Jean-Pierre , Gachet Christian
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Nuclear localization of protein kinase Cα and its association with nuclear components in Neuro‐2a neuroblastoma cells
Authors: Buchner Klaus , Lindschau Carsten , Hucho Ferdinand
Published: July 2002

Using activity measurements and Western blotting, we demonstrated that PKCα is constitutively present in nuclei of Neuro‐2a neuroblastoma cells. Confocal microscopy revealed that PKCα is present in the nucleoplasm and that this localization does not change after stimulation with phorbol ester. However, as revealed by extraction experiments, phorbol ester leads to a firmer association of PKCα with nuclear components. Our findings suggest that PKCα not only associates with lipids but also with proteins inside the nucleus. The presence of active PKCα inside the nucleus allows the enzyme to phosphorylate not only proteins at the nuclear envelope but also proteins in the nucleoplasm.
PHAS‐I phosphorylation in response to foetal bovine serum (FBS) is regulated by an ERK1/ERK2‐independent and rapamycin‐sensitive pathway in 3T3‐L1 adipocytes
Authors: Arnott Caroline H. , Atkinson Peter G.P. , Sale Elizabeth M. , Sale Graham J.
Published: July 2002

The phosphorylation state of PHAS‐I is thought to be important in the regulation of protein synthesis initiation. PHAS‐I phosphorylation significantly increases in response to growth factors and insulin. ERK1/ERK2 have previously been implicated as PHAS‐I kinases. Present work utilised a specific phosphorothioate oligonucleotide antisense strategy against ERK1/ERK2 to determine whether ERK1/ERK2 mediate FBS‐stimulated PHAS‐I phosphorylation in vivo. Depleting >90% of cellular ERK1/ERK2 had no effect on FBS‐stimulated PHAS‐I phosphorylation. However, treatment of cells with a specific p70S6k pathway inhibitor, rapamycin, markedly attenuated FBS‐stimulated PHAS‐I phosphorylation. These results indicate that PHAS‐I phosphorylation in response to FBS occurs through an ERK1/ERK2‐independent and rapamycin‐sensitive pathway in 3T3‐L1 adipocytes.
New insights on the functions of the guanylyl cyclase receptors
Authors: Wedel Barbara J , Garbers David L
Published: July 2002

The discovery of at least 29 genes encoding putative guanylyl cyclases in Caenorhabditis elegans has raised the question as to whether there are numerous receptors yet to be discovered in the mammal. The nematode, however, not only seems ideal to study guanylyl cyclase receptor localization and function, given the large variety of isoforms, but also leads to possible identification of ligands for orphan guanylyl cyclases by the use of genetic and behavioral assays. A recent powerful approach to describe the function of different guanylyl cyclase isoforms in mammals has been the disruption of the corresponding genes in the mouse. A salt resistant elevation of blood pressure, which corresponds to the phenotype of 50% of all human patients with essential hypertension, is observed in mice lacking the GC‐A‐receptor. Mice missing the GC‐C receptor have been shown to be resistant to STa, an E. coli heat‐stable enterotoxin, which is largely responsible for travellers diarrhea in adults and mortality due to diarrhea in infants.
Isolation and characterization of replication protein A (RP‐A) from tobacco cells
Authors: Garcia-Maya Mitla M , Buck Kenneth W
Published: July 2002

Replication protein A (RP‐A) was isolated from tobacco suspension cells and purified to near homogeneity by a procedure involving isolation of protoplasts, preparation of nuclei, nuclear lysis, binding to a column of single‐stranded (ss) DNA cellulose and elution at different salt concentrations. The purified protein contained three subunits with molecular masses of 70, 34 and 14 kDa, and was free from nuclease activity. Tobacco RP‐A had a high affinity for ssDNA. Binding competition experiments indicated only a weak affinity for double‐stranded DNA and no detectable affinity for ssRNA. Photochemical cross‐linking experiments indicated that the 70 kDa subunit has the ssDNA‐binding activity. Tobacco RP‐A was able to stimulate the activity of a tobacco α‐like DNA polymerase about 4‐fold. This is the first isolation of RP‐A from a plant and the procedure may be generally applicable to other plant species.
DNA repair activity in protein extracts from rat tissues
Authors: Coudoré François , Calsou Patrick , Salles Bernard
Published: July 2002

Among DNA repair pathways, nucleotide excision repair (NER) is able to recognize and process a wide variety of DNA lesions. The NER mechanism can be summarized in two stages: incision/excision of the lesion and DNA repair synthesis. Here, we have assessed the repair synthesis activity of protein extracts from different rat tissues by an in vitro biochemical assay that reproduces the entire NER reaction. The protein extraction procedure was adapted to rat tissues and the biochemical parameters of the assay (high salt concentration, addition of EGTA) in order to minimize non‐specific nuclease activity which allows the measurement of repair activity. Using this repair assay we detected a small increase in the extent of repair synthesis in liver compared to brain and lung tissue protein extracts. Similar results were obtained using a derivative assay that allows the measurement of the incision activity of tissue protein extracts with lower incision activity in lung tissue extract.
The different inhibitory domains of the Oct‐2 transcription factor have distinct functional activities
Authors: Gay Robert D. , Dawson Sally J. , Latchman David S.
Published: July 2002

The Oct‐2 POU family transcription factor contains three distinct regions whose deletion reduces its ability to inhibit transcription via its octamer binding site. Here we show that only one of these inhibitory domains is capable of also inhibiting the activity of activating molecules bound at adjacent sites upstream of a TATA box‐containing promoter whereas the other two regions are inactive in this assay. None of the three regions is able to achieve this effect when located upstream of the same promoter containing an initiator motif. The mechanisms of action of these domains and their role in the functioning of the Oct‐2 factor are discussed.
PPP1R6, a novel member of the family of glycogen‐targetting subunits of protein phosphatase 1
Authors: Armstrong Christopher G , Browne Gareth J , Cohen Philip , Cohen Patricia T.W
Published: July 2002

A complementary DNA encoding a novel human protein phosphatase 1 (PP1) glycogen‐targetting subunit of molecular mass 33 kDa has been sequenced. PPP1R6 is 31% identical to the glycogen‐targetting subunit (GL) of PP1 from rat liver, 28% identical to the N‐terminal region of the glycogen‐targetting subunit (GM) from human skeletal muscle and 27% identical to glycogen‐targetting subunit PPP1R5. Unlike human PPP1R5 and its murine homologue PTG, whose mRNAs are most abundant in skeletal muscle, heart and liver, PPP1R6 is present at similar levels in a wide variety of tissues. The PPP1R6 is associated with glycogen in muscle but is not subject to the same modes of covalent and allosteric regulation as GM and GL.
Effect of the human serum paraoxonase 55 and 192 genetic polymorphisms on the protection by high density lipoprotein against low density lipoprotein oxidative modification
Authors: Mackness Bharti , Mackness Michael I , Arrol Sharon , Turkie Wajdi , Durrington Paul N
Published: July 2002

Human serum paraoxonase (PON1) associated with high density lipoprotein (HDL) has been postulated to have a role in protecting low density lipoprotein (LDL) against oxidative modification, which has led to the proposal that PON1 is an anti‐atherogenic, anti‐inflammatory enzyme. PON1 has two genetically determined polymorphic sites giving rise to amino‐acid substitutions at positions 55 (L→M) and 192 (R→Q) and therefore 4 potential alloenzymes. We have examined the effects of these molecular polymorphisms on the ability of HDL to protect LDL from oxidative modification. HDL protected LDL from oxidative modification, whatever the combination of PON1 alloenzymes present in it. However, HDL from QQ/MM homozygotes was most effective at protecting LDL while HDL from RR/LL homozygotes was least effective. Thus after 6 h of co‐incubation of HDL and LDL with Cu2+PON1‐QQ HDL retained 57±6.3% of its original ability to protect LDL from oxidative modification, while PON1‐QR HDL retained less at 25.1±4.5% (P<0.01) and PON1‐RR HDL retained only 0.75±0.40% (P<0.005). In similar experiments HDL from LL and LM genotypes retained 21.8±7.5% and 29.5±6.6% (P=NS), respectively, of their protective ability, whereas PON1‐MM HDL maintained 49.5±5.3% (P<0.01). PON1 polymorphisms may affect the ability of HDL to impede the development of atherosclerosis and to prevent inflammation.
Substitution of Asn for Ser543 in the large fragment of Taq DNA polymerase increases the efficiency of synthesis of long DNA molecules
Authors: Ignatov K.B , Miroshnikov A.I , Kramarov V.M
Published: July 2002

Substitution of Asn for Ser543 in the large fragment of Taq DNA polymerase (Klentaq) increases several times the efficiency of synthesis of long (over 2 kbp) DNA molecules. The difference in the DNA synthesis efficiencies by the mutant and native enzymes increased with the increase in the DNA fragment length.
Inhibition of caspase activity induces a switch from apoptosis to necrosis
Authors: Lemaire Christophe , Andréau Karine , Souvannavong Vongthip , Adam Arlette
Published: July 2002

The role of caspases in B lymphocyte cell death was investigated by using two broad spectrum inhibitors of the caspase family, Z‐Asp‐cmk and Z‐VAD‐fmk. They totally prevented spontaneous and drug‐induced apoptosis and inhibited the CPP32/caspase‐3‐like activity exhibited by apoptotic cells. However, the suppression of apoptosis was not associated with a long‐term increase of cell survival, but conversely, with a switch from apoptotic death to the necrotic form. These results strongly suggest that apoptosis and necrosis share common initiation pathways, the final issue being determined by the presence of an active caspase.
Genetic engineering, production and characterisation of monomeric variants of the dimeric Serratia marcescens endonuclease
Authors: Franke Ingo , Meiss Gregor , Blecher Dinah , Gimadutdinow Oleg , Urbanke Claus , Pingoud Alfred
Published: July 2002

The Serratia nuclease is a non‐specific endonuclease which cleaves single‐ and double‐stranded RNA and DNA. It is a member of a large family of related endonucleases, most of which are dimers of identical subunits, with the notable exception of the Anabaena nuclease which is a monomer. In order to find out whether the dimer state of the Serratia nuclease is essential for its function we have produced variants of this nuclease which based on the crystal structure (Miller, M.D. and Krause, K.L. (1996), Protein Science 5, 24–33) were expected to be unable to dimerise. We demonstrate here that these variants, H184A, H184N, H184T and H184R, are monomers and have the same secondary structure, stability towards chemical denaturation and activity as the wild‐type enzyme. This allows to conclude that the dimeric state is not essential for the catalytic function of the Serratia nuclease. In contrast, the S179C variant which is also a monomer shows little activity, presumably because this amino acid substitution changes the structure of the enzyme.
Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol
Authors: Fellermeier Monika , Zenk Meinhart H
Published: July 2002

A new enzyme, geranylpyrophosphate:olivetolate geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.
Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1α: effect on α2A‐adrenoceptor‐Gi1α fusion protein activation
Authors: Carr I.Craig , Burt Andrew R , Jackson Vicky N , Wright Jason , Wise Alan , Rees Stephen , Milligan Graeme
Published: July 2002

Fusion proteins were constructed between the porcine α2A‐adrenoceptor and either wild‐type (Cys351) or a pertussis toxin‐resistant (Gly351) form of the G protein Gi1α. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration‐dependent stimulation of their high affinity GTPase activity. The α2A‐adrenoceptor‐wild type Gi1α fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly351 Gi1α. Treatment of the fusion proteins as agonist‐regulated enzymes allowed measurement of V
max and turnover number for adrenaline‐stimulation of the GTPase activity of each fusion construct. The turnover number of the α2A‐adrenoceptor‐Cys351Gly Gi1α fusion protein was only 44% of that for the α2A‐adrenoceptor‐wild type Gi1α fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist‐occupied receptor to activate the mutant.
Skeletal muscle contractions stimulate cGMP formation and attenuate vascular smooth muscle myosin phosphorylation via nitric oxide
Authors: Lau Kim S. , Grange Robert W. , Chang Wen-Jinn , Kamm Kristine E. , Sarelius Ingrid , Stull James T.
Published: July 2002

Nitric oxide generated by neuronal nitric oxide synthase in contracting skeletal muscle fibers may regulate vascular relaxation via a cGMP‐mediated pathway. Neuronal nitric oxide synthase content is greatly reduced in skeletal muscles from mdx mice. cGMP formation increased in contracting extensor digitorum longus muscles in vitro from C57 control, but not mdx mice. The increase in cGMP content was abolished with N
G‐nitro‐l‐arginine. Sodium nitroprusside treatment increased cGMP levels in muscles from both C57 and mdx mice. Skeletal muscle contractions also inhibited phenylephrine‐induced phosphorylation of smooth muscle myosin regulatory light chain. Arteriolar dilation was attenuated in contracting muscles from mdx but not C57 mice. NO generated in contracting skeletal muscle may contribute to vasodilation in response to exercise.
Secondary structure of the membrane‐bound domains of H+,K+‐ATPase and Ca2+‐ATPase, a comparison by FTIR after proteolysis treatment of the native membranes
Authors: Raussens Vincent , le Maire Marc , Ruysschaert Jean-Marie , Goormaghtigh Erik
Published: July 2002

The sarcoplasmic reticulum Ca2+‐ATPase and the gastric H+,K+‐ATPase were cleaved under three different proteolysis conditions. After elimination of the protease and of the cleaved peptides, the vesicles containing the membrane‐bound peptides of the ATPases were studied by Fourier transform attenuated total reflection infrared spectroscopy. In the harsher proteolysis conditions, the membrane‐associated domain of the Ca2+‐ATPase represented about 20% of the protein and was mainly constituted of α‐helices. Polarized infrared spectroscopy showed that these α‐helices were mainly oriented perpendicular to the membrane. However, only 10–20% of the H+,K+‐ATPase was cleaved. The remaining, membrane‐associated domain of the protein contained about 30% of α‐helices and 30% of β‐sheet structures. The α‐helices adopted a mainly transmembrane orientation. While the data on the Ca2+‐ATPase are in general agreement with the current model of the protein, our results indicate that caution must be used in choosing this protein as a general structural model for all P‐type ATPases. The protease‐resistant, membrane‐associated domain of the H+K+‐ATPase is indeed much larger than predicted and also contained β‐sheet structures.
The Na+ and K+ transport deficiency of an E. coli mutant lacking the NhaA and NhaB proteins is apparent and caused by impaired osmoregulation
Authors: Verkhovskaya Marina L , Barquera Blanca , Verkhovsky Michael I , Wikström Mårten
Published: July 2002

Cells of the E. coli mutant EP432, which lacks the two Na+/H+ antiporters, NhaA and NhaB, have been reported to have an impaired sodium transport activity (Harel‐Bronstein et al. (1995) J. Biol. Chem. 270, 3816–3822). Here we report that active transport of Na+ in EP432 cells can be restored to wild‐type levels, either by a high K+ concentration or by an increase in the medium osmolarity. We suggest that this mutant is primarily deficient in osmoregulation rather than in cation transport per se.
Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase
Authors: Gudima S.O , Kostyuk D.A , Grishchenko O.I , Tunitskaya V.L , Memelova L.V , Kochetkov S.N
Published: July 2002

Synthesis of deoxynucleotide‐containing RNA‐like single‐stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter‐containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8–10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1–+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non‐coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG‐containing) was achieved with high yields.
Identification and characterization of N‐acetylglucosamine‐6‐O‐sulfate‐specific β1,4‐galactosyltransferase in human colorectal mucosa
Authors: Seko Akira , Hara-Kuge Sayuri , Yonezawa Suguru , Nagata Koji , Yamashita Katsuko
Published: July 2002

6‐Sulfo‐sialyl Lewis X structure is attributable to recognition between lymphocytes and high endothelial venules. However, the biosynthetic pathway still remains unclear. We found that a β‐galactosyltransferase (βGalT) in human colorectal mucosa preferentially acts on GlcNAc‐6‐O‐sulfate (6S‐GN). 6S‐GN:β4GalT was partially purified by UDP‐hexanolamine‐Sepharose and asialo‐agalacto‐ovomucin‐Sepharose chromatographies. The optimum pH of this enzyme was found to be 6.5–7.5 and the Michaelis constants for 6S‐GN and UDP‐Gal were 0.43 mM and 16 μM, respectively. The enzymatic activity was dependent on divalent cations and the substrate specificity was not affected by α‐lactalbumin. This is the first demonstration of the occurrence of 6S‐GN:β4GalT.
Reactivity of glutaredoxins 1, 2 and 3 from Escherichia coli and protein disulfide isomerase towards glutathionyl‐mixed disulfides in ribonuclease A
Authors: Lundström-Ljung Johanna , Vlamis-Gardikas Alexios , Åslund Fredrik , Holmgren Arne
Published: July 2002

We have examined the activity of protein disulfide isomerase (PDI) and glutaredoxin (Grx) 1, 2 and 3 from Escherichia coli to catalyze the cleavage of glutathionylated ribonuclease A (RNase‐SG) by 1 mM GSH to yield reduced RNase. Apparent K
m values for RNase‐SG were similar, 2–10 μM, for Grx 1, 3 and PDI but Grx 1 and Grx 3 showed 500‐fold higher turnover numbers than PDI. The atypical Grx 2 also catalyzed deglutathionylation by GSH, but had higher K
m and apparent turnover number values compared to the two classical Grx. Refolding of RNase in a glutathione redox buffer was catalyzed by PDI. However, it could be measured only after a characteristic lag phase that was shortened by all three E. coli Grxs in a concentration‐dependent manner. A role of the glutaredoxin mechanism in the endoplasmic reticulum is suggested.
The vacuolar Ca2+/H+ exchanger Vcx1p/Hum1p tightly controls cytosolic Ca2+ levels in S. cerevisiae
Authors: Miseta Attila , Kellermayer Richard , Aiello David P , Fu Lianwu , Bedwell David M
Published: July 2002

It is well established that the vacuole plays an important role in the cellular adaptation to growth in the presence of elevated extracellular Ca2+ concentrations in Saccharomyces cerevisiae. The Ca2+ ATPase Pmc1p and the Ca2+/H+ exchanger Vcx1p/Hum1p have been shown to facilitate Ca2+ sequestration into the vacuole. However, the distinct physiological roles of these two vacuolar Ca2+ transporters remain uncertain. Here we show that Vcx1p can rapidly sequester a sudden pulse of cytosolic Ca2+ into the vacuole, while Pmc1p carries out this function much less efficiently. This finding is consistent with the postulated role of Vcx1p as a high capacity, low affinity Ca2+ transporter and suggests that Vcx1p may act to attenuate the propagation of Ca2+ signals in this organism.
The structure of chromatophores from purple photosynthetic bacteria fused with lipid‐impregnated collodion films determined by near‐field scanning optical microscopy
Authors: Shinkarev Vladimir P , Brunner Robert , White Jeffrey O , Wraight Colin A
Published: July 2002

Lipid‐impregnated collodion (nitrocellulose) films have been frequently used as a fusion substrate in the measurement and analysis of electrogenic activity in biological membranes and proteoliposomes. While the method of fusion of biological membranes or proteoliposomes with such films has found a wide application, little is known about the structures formed after the fusion. Yet, knowledge of this structure is important for the interpretation of the measured electric potential. To characterize structures formed after fusion of membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides with lipid‐impregnated collodion films, we used near‐field scanning optical microscopy. It is shown here that structures formed from chromatophores on the collodion film can be distinguished from the lipid‐impregnated background by measuring the fluorescence originating either from endogenous fluorophores of the chromatophores or from fluorescent dyes trapped inside the chromatophores. The structures formed after fusion of chromatophores to the collodion film look like isolated (or sometimes aggregated, depending on the conditions) blisters, with diameters ranging from 0.3 to 10 μm (average ≈1 μm) and heights from 0.01 to 1 μm (average ≈0.03 μm). These large sizes indicate that the blisters are formed by the fusion of many chromatophores. Results with dyes trapped inside chromatophores reveal that chromatophores fused with lipid‐impregnated films retain a distinct internal water phase.
Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen
Authors: Mogi Tatsushi , Sato-Watanabe Mariko , Miyoshi Hideto , Orii Yutaka
Published: July 2002

To probe the functional role of a bound ubiquinone‐8 in cytochrome bo‐type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol‐1 and dioxygen. Stopped‐flow studies showed that anaerobic reduction of the wild‐type and the bound ubiquinone‐free (ΔUbiA) enzymes with ubiquinol‐1 immediately takes place with four kinetic phases. Replacement of the bound ubiquinone with 2,6‐dibromo‐4‐cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol‐1 by eliminating the fast phase. Flow‐flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b to heme o electron transfer occurs with a rate constant of ∼104 s−1 in all three preparations. These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low‐spin heme b in subunit I.
Two highly conserved glutamic acid residues in the predicted β propeller domain of dipeptidyl peptidase IV are required for its enzyme activity
Authors: Abbott Catherine A , McCaughan Geoffrey W , Gorrell Mark D
Published: July 2002

Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N‐terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp‐Trp‐(Val/Ile/Leu)‐Tyr‐Glu‐Glu‐Glu (DW(V/I/L)YEEE) in the predicted β propeller domain of the DPP IV‐like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser‐Asp‐His catalytic triad.
Expression of the AMP‐activated protein kinase β1 and β2 subunits in skeletal muscle
Authors: Chen Zhi-ping , Heierhorst Jörg , Mann Richard J. , Mitchelhill Ken I. , Michell Belinda J. , Witters Lee A. , Lynch Gordon S. , Kemp Bruce E. , Stapleton David
Published: July 2002

A heterotrimeric member of the AMP‐activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an α2 catalytic and two non‐catalytic subunits, β2 and γ1. The AMPK β2 cDNA (271 amino acids (aa), molecular weight (MW)=30 307, pI 6.3) was cloned from skeletal muscle and found to share an overall identity of 70% with β1 (270 aa, MW=30 475, pI 6.0). In the liver AMPK β1 subunit, Ser‐182 is constitutively phosphorylated whereas in skeletal muscle β2 isoform, we find that Ser‐182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser‐24, Ser‐25 found in the β1 are replaced by Ala‐Glu in the β2 isoform. β2 contains seven more Ser and one less Thr residues than β1, raising the possibility of differential post‐translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively β1 associated with α2, whereas extensor digitorum longus muscle α2 (EDL, fast twitch) associates with β2 as well as β1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co‐immunoprecipitated with the AMPK α2β2γ1 complex.
Glucose metabolism in cholinoceptive cortical ratbrain regions after basal forebrain cholinergic lesion
Authors: Mehlhorn Gaby , Löffler Thomas , Apelt Jenny , Robner Steffen , Urabe Takao , Hattori Nobutaka , Nagamatsu Shinya , Bigl Volker , Schliebs Reinhard
Published: July 2002

To address the question whether the changes in cortical glucose metabolism observedin patients with Alzheimers disease are interrelated with, or consequences of, basal forebraincholinergic cell loss, an experimental approach was employed to produce cortical cholinergicdysfunction in rat brain by administration of the cholinergic immunotoxin 192IgG‐saporin. [14C]d‐glucose utilization in brain homogenates, d‐glucose‐displaceable [3H]cytochalasin B binding to glucose transporters (GLUT),Northern and Western analyses, as well as in vivo [14]2‐deoxyglucoseautoradiography were used to quantify the regional glucose metabolism.

Basal forebrain cholinergic lesion resulted in transient increases in glucose transporterbinding in cortical regions displaying reduced acetylcholinesterase activity, already detectableseven days after lesion with peak values around 30 days post lesion. Western analysis revealedthat the changes in total glucose transporter binding are mainly due to changes in the GLUT3subtype only, while the levels of GLUT1 and GLUT3 mRNA (Northern analysis) were notaffected by cholinergic lesion. Both immunocytochemistry and in situ hybridizationdemonstrated preferential localizations of GLUT1 on brain capillaries and GLUT3 on neurons,respectively. A lesion‐induced transient decrease in [14C]d‐glucoseutilization seven days post lesion was detected in the lesion site, whereas cholinoceptive corticalregions were not affected. In vivo [14C]deoxyglucose uptake was transientlyincreased in cholinoceptive cortical regions and in the lesion site being highest between three toseven days after lesion.

The cholinergic lesion‐induced transient up‐regulation of cortical glucose transporters anddeoxyglucose uptake reflects an increased glucose demand in regions depleted by acetylcholinesuggesting functional links between cortical cholinergic activity and glucose metabolism incholinoceptive target regions.
The role of protein kinase Cα in U‐87 glioma invasion
Authors: Cho Kyung‐Keun , Mikkelsen Tom , Lee Yong J. , Jiang Feng , Chopp Michael , Rosenblum Mark L.
Published: July 2002

To investigate the hypothesis that protein kinase Cα (PKCα) is functional glial tumor cell invasion, stable PKCα sense and antisense transfected U‐87 cell lines were established and PKCα expression characterized by Western blot and PKC activity assays. Invasion assays including barrier migration (Koochekpour et al., Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma lines. Eur. J. Cancer,1995, 31, 375–380; Merzak et al., CD44 mediates human glioma cell adhesion and invasion in vitro. Cancer Res., 1994, 54, 3988–3992; Merzak et al., Cell surface gangliosides are involved in the control of human glioma cell invasion in vitro. Neurosci. Lett., 1994, 177, 11–16), and spheroid confrontation were used to study the relationship between PKCα expression and invasiveness. PKCα overexpressing clones show increased barrier migration (1.5×) relative to the control transfected clones. PKCα inhibited clones exhibited reduced invasiveness, to <50%. In coculture with PKCα overexpressing clones, the remaining normal fetal rat brain aggregate volume was significantly decreased (up to 20%) but 90% of the initial brain volume was left in PKCα inhibited clone in the rat brain aggregate tumor spheroid confrontation. This effect was not associated with significant growth inhibition. We conclude that expression of PKCα in glioma‐derived cell lines appears to be central to glioma invasion in vitro.
Clinical features of panic attacks in schizophrenia
Authors: Baylé F.J. , Krebs M.O. , Epelbaum C. , Levy D. , Hardy P.
Published: July 2002

Since reports have underscored that panic attacks (PA) may be an identifiable state occurring in schizophrenia, we studied the symptomatology of PA in a group of schizophrenic patients. Of 40 patients (21 males and 19 females) attending a clinic for maintenance therapy of schizophrenia, 19 (36.8%) had a lifetime history of PA. Seven among those 19 patients (36.8%) had or had had spontaneous panic attacks, not related to phobic fears or delusional fears, and for the 12 remaining patients, the PA were related to paranoid ideas. Moreover, the paranoid subtype of schizophrenia tends to be more often associated with a history of panic attack than other subtypes of schizophrenia (52.6% vs 23.8%; χ2 = 3.5, P = .06). It seems that there are at least two types of PA in schizophrenic patients. The first one could be independent from the psychotic feature, with no psychopathological link. The second kind of PA could be directly related to a schizophrenic disorder, and found in patients with the paranoid subtype.
Methodological issues in the design of clinical studies of new drugs for
the treatment of obsessive compulsive disorder
Authors: Holland RL
Published: July 2002

Obsessive Compulsive Disorder (OCD) is increasingly recognised to be
relatively common and responsive to some drug treatments (serotonin reuptake
inhibitors). Clinical studies utilising recognised diagnostic criteria have
identified some relevant negative prognostic factors: early age at disease
onset, severe baseline disease, and failure of previous drug treatments all
of which reduce the apparent success rates of SRI drugs. Furthermore,
patients with comorbid tics seem to respond best to a combination of SRI
activity and dopamine blockade. Short term efficacy studies are best
performed with a parallel group design and a duration of around 3 months.
Long term efficacy studies should probably use the relapse prevention
design, with open label responders randomised to double blind continuation
or placebo. Does finding has not been properly explored, and any one fixed
dose will only be optimal for some patients. Efficacy can be reliably
measured using the YBOCs, NIMH-OC and CGI rating scales.
Economics of chronic heart failure
Authors: Berry Colin , Murdoch David R. , McMurray John J.V.
Published: July 2002

Chronic heart failure (CHF) is now recognized as a major and escalating public health problem. The costs of this syndrome, both in economic and personal terms, are considerable. The prevalence of CHF is 1–2% and appears to be increasing, in part because of ageing of the population. Economic analyses of CHF should include both direct and indirect costs of care. Healthcare expenditure on CHF in developed countries consumes 1–2% of the total health care budget. The cost of hospitalization represents the greatest proportion of total expenditure. Optimization of drug therapy represents the most effective way of reducing costs. Recent economic analyses in the Netherlands and Sweden suggest the costs of care are rising.
Early estimation soil‐cement for enlarged bases in bored precast piling method based on cement‐water ratio
Authors: Yoda Yuya , Asaka Yoshiharu , Yamato Yuka , Kimura Takumi , Kishida Satoru
Published: May 2020

The bored precast piling method is used to obtain bearing loads via construction of an enlarged base and a pile fixture part by injecting and agitating cement milk into mud in an excavated hole, whereby the pile tip reaches the bearing stratum. Achieving the requisite strength of the soil‐cement mix, from which the enlarged base is constructed, is critical for ensuring that the bearing load is withstood. In this study, several aspects of this piling method were investigated to assess its applicability to a wide range of soil types and to construct a rapid and highly accurate quality control method. First, the material composition of the soil‐cement mix used in the enlarged base of a bored precast pile was investigated. Next, the parameters affecting the evaluation of the strength of the soil‐cement mix were identified via laboratory mixing tests, and an equation for strength estimation was established. Finally, a method was proposed for analyzing the material composition of the soil‐cement mix at the site, and technology was developed to enable early estimation of the 28‐ day(s) compressive strength based on the material composition. Ultimately, the estimation accuracy of this methodology was verified at 80 sites.
Commissioning of a 1.5T Elekta Unity MR‐linac: A single institution experience
Authors: Snyder Jeffrey E. , St‐Aubin Joël , Yaddanapudi Sridhar , Boczkowski Amanda , Dunkerley David A.P. , Graves Stephen A. , Hyer Daniel E.
Published: May 2020

MR image‐guided radiotherapy has the potential to improve patient care, but integration of an MRI scanner with a linear accelerator adds complexity to the commissioning process. This work describes a single institution experience of commissioning an Elekta Unity MR‐linac, including mechanical testing, MRI scanner commissioning, and dosimetric validation. Mechanical testing included multileaf collimator (MLC) positional accuracy, measurement of radiation isocenter diameter, and MR‐to‐MV coincidence. Key MRI tests included magnetic field homogeneity, geometric accuracy, image quality, and the accuracy of navigator‐triggered imaging for motion management. Dosimetric validation consisted of comparison between measured and calculated PDDs and profiles, IMRT measurements, and end‐to‐end testing. Multileaf collimator positional accuracy was within 1.0 mm, the measured radiation isocenter walkout was 0.20 mm, and the coincidence between MR and MV isocenter was 1.06 mm, which is accounted for in the treatment planning system (TPS). For a 350‐mm‐diameter spherical volume, the peak‐to‐peak deviation of the magnetic field homogeneity was 4.44 ppm and the geometric distortion was 0.8 mm. All image quality metrics were within ACR recommendations. Navigator‐triggered images showed a maximum deviation of 0.42, 0.75, and 3.0 mm in the target centroid location compared to the stationary target for a 20 mm motion at 10, 15, and 20 breaths per minute, respectively. TPS‐calculated PDDs and profiles showed excellent agreement with measurement. The gamma passing rate for IMRT plans was 98.4 ± 1.1% (3%/ 2 mm) and end‐to‐end testing of adapted plans showed agreement within 0.4% between ion‐chamber measurement and TPS calculation. All credentialing criteria were satisfied in an independent end‐to‐end test using an IROC MRgRT phantom.
Prolonged confusional state as first manifestation of COVID‐19
Authors: Butt Isabel , Sawlani Vijay , Geberhiwot Tarekegn
Published: May 2020

A 77‐year‐old gentleman, normally fit and well, was admitted with acute confusion. On admission, Glasgow Coma Scale (GCS) was 14/15, vital signs were within the normal limits and bilateral crepitation at the lung base. Head CT scan was normal. CXR showed some air space opacification. Investigations revealed hyponatraemia, raised CRP, and positive for COVID‐19. Treated with antibiotics and intravenous saline, sodium returned to normal. Delirium remained unchanged 4 weeks post‐incidence. Neurological manifestations were documented in patients with COVID‐19; however no report has shown delirium as a primary manifestation. This case illustrates acute confusion may be the only presenting symptom of COVID‐19 without overt lung disease.
Preeclampsia Risk Prediction Model for Chinese Pregnant Patients With Systemic Lupus Erythematosus
Authors: Jiang Meng , Wang You , Fu Qiong , Lin Sihan , Wu Jiayue , Di Wen
Published: May 2020

Objective

To screen for a high risk of preeclampsia in women with systemic lupus erythematosus (SLE).

Methods

A total of 513 antenatal care records of pregnant patients with SLE were obtained, and the data were randomly assigned to either a development set (n = 342) or a validation set (n = 171). Preeclampsia predictors were identified with stepwise regression, and a coefficient B of each variable was used to establish a prediction model and risk scoring system. Goodness‐of‐fit was assessed by the Hosmer‐Lemeshow and Omnibus tests, and the area under the receiver operating characteristic curve (area under the curve) was used to assess discrimination. Validation was performed using the validation set.

Results

The preeclampsia incidence was 14.4% in the pregnant patients with SLE. A mean arterial pressure (MAP) ≥96.5 mm Hg (odds ratio [OR] 213.15 [95% confidence interval (95% CI) 24.39–999.99]), prepregnancy hypertension (OR 18.19 [95% CI 2.67–125.01]), a hematologic disorder (OR 4.13 [95% CI 1.03–16.67]), positive IgM anticardiolipin antibodies (aCLs) (OR 19.85 [95% CI 1.11–333.33]), serum albumin <31.5 grams/liter (OR 9.88 [95% CI 2.07–47.62]), serum uric acid ≥303 μmoles/liter (OR 5.58 [95% CI 1.40–22.22]), and 24‐hour urinary protein ≥0.286 grams (OR 14.39 [95% CI 2.43–83.33]) were selected for the preeclampsia prediction model. The area under the curve was 0.975. Preeclampsia prediction model scores >4 indicated a high risk of preeclampsia. For the validation set, the preeclampsia prediction accuracy was 93.6% (sensitivity 88.5%, specificity 94.5%).

Conclusion

A model for predicting the risk of preeclampsia in pregnant patients with SLE was established on the basis of MAP, prepregnancy hypertension, hematologic disorders, IgM aCLs, albumin, uric acid, and 24‐hour urinary protein. The model had good predictive efficiency and can help clinicians improve pregnancy outcomes in high‐risk women with early interventions.
Correlations to Predict Microstructure and Mechanical Properties of Ultrasonically Cast Metal Matrix Nanocomposites as a Function of Treatment Time
Authors: Paul Tanaji , Zhang Cheng , Boesl Benjamin , Agarwal Arvind
Published: May 2020

A quantitative understanding of the role of ultrasonic treatment (UST) process variables on microstructure and mechanical properties is critical for the development of process maps for manufacturing metal matrix composites (MMCs). This article presents a novel mathematical framework to delineate the functional correlations among ultrasonication time, grain refinement, and hardening in SiC nanoparticle‐reinforced Al matrix composites. UST generates microbubbles and deagglomerates SiC to increase heterogeneous nucleation sites synergistically. The increase in volumetric nucleation density due to SiC addition exhibits slow exponential kinetics with varying ultrasonication time. An outstanding grain refinement efficiency of 62.8% is achieved upon ultrasonication for 90 s. The contributions to an increase in the hardness due to grain refinement and SiC dispersion are isolated to develop correlations between ultrasonication time and hardening. Hardening increases exponentially with treatment time due to the reduction of interparticle distance from sonication‐induced SiC dispersion. These fundamental mathematical correlations constitute a significant advancement toward the development of ultrasonic process maps and MMC manufacturing technology.
Pseudocapacitive Trimetal Fe0.8CoMnO4 Nanoparticles@Carbon Nanofibers as High‐Performance Sodium Storage Anode with Self‐Supported Mechanism
Authors: Wu Lijun , Yang Jinyu Ou , Guo Shoujie , Yao Liangxian , Li Hao , Zhang Shixing , Yue Hongwei , Cai Kun , Zhang Chao , Yang Changchun , Cao Yuliang
Published: May 2020

Trimetal Fe0.8CoMnO4 (FCMO) nanocrystals with a diameter of about 50 nm perfectly embedded in N doped‐carbon composite nanofibers (denoted as FCMO@C) are successfully prepared through integrating double‐nozzle electrospinning with a drying and calcination process. The as‐prepared FCMO@C nanofibers maintain a high reversible capacity of 420 mAh g−1 and about 90% capacity retention after 200 cycles at 0.1 A g−1. For a long‐term cycle, the FCMO@C electrode exhibits excellent cycling stability (87% high capacity retention at 1 A g−1 after 950 cycles). Kinetic analysis demonstrates that the electrochemical characteristics of the FCMO@C corresponds to the pseudocapacitive approach in charge storage as an anode for sodium ion batteries, which dominantly attributes the credit to FCMO nanocrystals to shorten the migration distance of Na+ ions and the nitrogen‐doped carbon skeleton to enhance the electronic transmission and favorably depress the volume expansion during the repeated insertion/extraction of Na+ ions. More significantly, a self‐supported mechanism via continuous electrochemical redox reaction of Fe, Co, and Mn can effectively relieve the volume change during charge and discharge. Therefore, this work can provide a new avenue to improve the sodium storage performance of the oxide anode materials.
Layered Silicon‐Based Nanosheets as Electrode for 4 V High‐Performance Supercapacitor
Authors: Gao Runsheng , Tang Jie , Yu Xiaoliang , Lin Shiqi , Zhang Kun , Qin Lu‐Chang
Published: May 2020

Silicon‐based materials have shown great potential and been widely studied in various fields. Unlike its unparalleled theoretical capacity as anodes for batteries, few investigations have been reported on silicon‐based materials for applications in supercapacitors. Here, an electrode composed of layered silicon‐based nanosheets, obtained through oxidation and exfoliation, for a supercapacitor operated up to 4 V is reported. These silicon‐based nanosheets show an areal specific capacitance of 4.43 mF cm−2 at 10 mV s−1 while still retaining a specific capacitance of 834 µF cm−2 even at an ultrahigh scan rate of 50 000 mV s−1. The volumetric energy and power density of the supercapacitor are 7.65 mWh cm−3 and 9312 mW cm−3, respectively, and the electrode can operate for 12000 cycles in a potential window of 4 V at 2 A g−1, while retaining 90.6% capacitance. These results indicate that the silicon‐based nanosheets can be a competitive candidate as the supercapacitor electrode material.