bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate and inhibitor studies on proteinase 3
Authors: Kam Chih-Min , Kerrigan John E. , Dolman Koert M. , Goldschmeding Roel , Von dem Borne Albert E.G.Kr. , Powers James C.
Published: July 2002

Various amino acid and peptide thioesters were tested as substrates for human proteinase 3 and the best substrate is Boc‐Ala‐Ala‐Nva‐SBzl with a k
cal/K
m value of 1.0 × 106nMit‐1sit‐1 Boc‐Ala‐Ala‐AA‐SBzl (AA = Val, Ala, or Met) are also good substrates with k
cal/K
m values of (1‐4) × 105 M−1 s−1. Substituted isocoumarins are potent inhibitors of proteinase 3 and the best inhibitors are 7‐amino‐4‐chloro‐3‐(2‐bromoethoxy)isocoumarin and 3.4‐dichloroisocoumarin (DCI) with k
obs/[I] values of 4700 and 2600 M−1 s−1, respectively. Substituted isocoumarins. peptide phosphonates and chloromethyl ketones inhibited proteinase 3 less potently than human neutrophil elastase (HNE) by 1‐2 orders of magnitude.
Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis Kinetic and spectroscopic evidence
Authors: Coletta Massimo , Ascenzi Paolo , Smulevich Giulietta , Mantini Anna Rita , Gaudio Rosanna Del , Piscopo Marina , Geraci Giuseppe
Published: July 2002

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.O. Protonation of the proximal imidazole at low pH (≤ 3.0) and the consequent cleavage of the HisF8NE2‐Fe proximal bond brings about a ≈ 20‐fold increase of the second‐order rate constant for CO binding. This process displays a pK
a = 4.0 ± 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2‐Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH‐rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828–4830), can be described only by assuming a concerted proton‐linked transition with n = 1.8 ± 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.
The amino acid sequence of a 20 kDa bifunctional subtilisin/α‐amylase inhibitor from brain of rice (Oryza saliva L.) seeds
Authors: Ohtsubo Ken-Ichi , Richardson Michael
Published: July 2002

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the α‐amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion‐exchange chromatography on DEAE‐Cellulose and Toyopearl CM‐650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S‐alkylated protein, and by manual DABITC/PITC micro‐sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, clastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.
The membrane insertion of colicins
Authors: Lakey J.H. , González-Mañas J.M. , van der Goot F.G. , Pattus F.
Published: July 2002

Pore‐forming toxins, such as colicin A, are water‐soluble proteins that insert into lipid bilayers. The water‐soluble structure of Colicin A is known at a high resolution and this review describes the kinetic and structural steps involved in its soluble‐to‐membrane bound transformation.
Molluscan putative prohormore convertases: Structural diversity in the central nervous system of Lymnaea stagnalis
Authors: Smit August B. , Spijker Sabine , Geraerts Wijnand P.M.
Published: July 2002

In the cerebral ganglia of the central nervous system of the freshwater snail Lymnaea stagnalis many neuropeptides are proteolytically processed from larger prohormones at sites marked by both single and multiple basic amino acids. In the present study we identified cloned cDNA and PCR products corresponding to three putative endoproteases that may be involved in prohormone processing. The cDNA encodes a protein of 653 residues with an overall sequence identity of 60%, 41%, 35%, 40%, and 27% with the recently characterized endoproteases PC2, PC1/3, PC4, furin and Kex2, respectively. The Lymnaea preproconvertase has ∼80% homology with the catalytic domain, and ∼40% and 5̃0 with the N‐and C‐terminal part, respectively, of the vertebrate PC2. Two cloned PCR products, Lfur 1 and Lfur 2, show highest sequence identity to furin. Expression of the LPC2 gene is exclusively in the central nervous system, where two LPC2 transcripts of 3.0 and 4.8 kb were detected.
Spontaneous thioester bond formation in α2‐macroglobulin, C3 and C4
Authors: Pangburn Michael K.
Published: July 2002

Purified α2‐macroglobulin and complement proteins C3 and C4 were treated with a ammonia to break their intramolecular thioester bonds and reform the original free cysteinyl and glutamyl side chains. When this reaction was performed at low temperature a conformational intermediate was trapped which lacked a thioester, but which could refold to the native structure and spontaneously reform the thioester and full biological function. The findings suggest that these proteins may undergo spontaneous post‐translational self‐modification forming the thioesters without involvement of enzymes of high energy metabolites such as ATP.
Inhibition of epidermal growth factor receptor functions by tyrosine kinase inhibitors in NIH3T3 cells
Authors: Umezawa Kazuo , Sugata Daiji , Yamashita Katsuyuki , Johtoh Noriko , Shibuya Masabumi
Published: July 2002

Epidermal growth factor (EGF) induces transformed phenotypes in EGF receptor‐overexpressing NIH3T3 (ER12) cells. Tyrosine kinase inhibitors such as erbstatin and its stable analogue methyl 2,5‐dihydroxycinnamate inhibited the EGF‐induced phenotypes changes in these cells; while 5′‐O‐methylerbstatin, an inactive analogue, did not. Methyl 2,5‐dihydroxycinnamate inhibited intracellular tyrosine kinase activity in EGF‐treated ER12 cells. Methyl 2,5‐dihydroxycinnamate also delayed the EGF‐induced DNA synthesis from the quiescent phase ER12 cells without showing irreversible cytotoxicity. It inhibited the DNA synthesis most efficiently at the early G1 phase. Thus, tyrosine kinase inhibitors may modify malignant phenotypes in EGF receptor‐overexpressing neoplasms.
Adenosine stimulates 5'‐nucleotidase activity in rat mesangial cells via A2 receptors
Authors: Stefanovic V. , Vlahovic P. , Savic V. , Ardaillou N. , Ardaillou R.
Published: July 2002

Because A2 adenosine receptor activation stimulates adenylate cyclase and cyclic AMP induces 5'‐nucleotidase expression in rat mesangial cells, we examined the effect of adenosine and its analogs on 5'‐nucleotidase activity in these cells. A2 adenosine receptors were characterized using [3H]5'‐N‐ethylcarboxamidoadenosine (NECA) as a tracer. There was a single group of receptor sites with a K
D value of 0.53 μM and a number of sites of 1,317 fmol/mg. [3H]NECA binding was inhibited preferentially by A2 adenosine analogs and antagonists. Similarly, the order of potency for cAMP stimulation was in favour of A2 adenosine analogs. Rat mesangial cells expressed surface 5'‐nucleotidase activity. Exposure of cells for 48 h to adenosine analogs showed that at low concentrations A2 analogs stimulated 5'‐nucleotidase activity. These results indicate that adenosine upregulates activity of 5'‐nucleotidase, the enzyme responsible for its local formation, via A2 receptor stimulation and increase in cAMP production.
Changed lateral migration of phospho‐LHCII in the thylakoid membrane upon acclimation of spinach to low temperatures
Authors: Carlberg Inger , Andersson Bertil
Published: July 2002

Movement of proteins along the plant thylakoid membrane is of importance for several physiological events, such as state transitions and turnover and repair of the photosystem II complex. Such lateral migrations are impaired at low temperatures, which could contribute to the increased sensitivity of plants to photoinhibitory damages at low temperatures. The migration behaviour ofphospho‐LHCII in thylakoid membranes isolated from cold‐acclimated spinach was studied and compared to that in control membranes. The rate of migration of phospho‐LHCII at low temperatures is increased 2‐ to 3‐fold and the apparent activation energy of the migration is decreased after the cold acclimation.
27‐Hydroxycholesterol modulation of low density lipoprotein metabolism in cultured human hepatic and extrahepatic cells
Authors: Bellosta Stefane , Corsini Alberto , Bernini Franco , Granata Agnese , Didoni Guido , Mazzotti Maria , Fumagalli Remo
Published: July 2002

27‐Hydroxycholesterol, 25‐hydroxycholesterol and cholesterol suppressed LDL uptake and degradation in human extrahepatic and hepatic cell lines hi a concentration‐dependent manner. Cholesterol was the least potent, and the inhibitory effect of oxysterols was more pronounced in skin fibroblasts and in endothelial cell line EAhy 926 than in hepatoma HepG2 cells. Shorter incubations were required for oxysterols to achieve 50% inhibition of LDL uptake and degradation in all cultured cells. The inhibition of LDL catabolism in extrahepatic cells by 27‐hydroxycholesterol occurred at concentrations close to those observed in human plasma (0.2–0.6 μM). The results support a possible role of 27‐hydroxycholesterol, a physiological oxysterol, in the regulation of cellular cholesterol homeostasis in non‐hepatic tissues.
Insulin activates myelin basic protein (p42 MAP) kinase by a protein kinase C‐independent pathway in rat adipocytes
Authors: Yang YunChung , Farese Robert V.
Published: July 2002

Myelin basic protein kinase (MBPK) activity of rat adipocytes was measured directly or in gels after purification of p42 microtubule‐associated protein kinase (MAPK). Insulin and phorbol esters provoked 2‐ to 3‐fold increases in MBPK/MAPK activity within 5–10 min. Whereas phorbol ester effects were blocked by protein kinase C (PKC) depletion or inhibition, insulin effects were fully intact, indicating that insulin activates MBPK/MAPK independently of PKC. In contrast, PKC depletion or inhibition markedly inhibited insulin effects on [3H]2‐deoxyglucose uptake, suggesting that this effect requires PKC, rather than a factor within the ras/MAPK cascade.
α‐Latrotoxin receptor
Authors: Petrenko Alexander G.
Published: July 2002

α‐Latrotoxin is a potent stimulator of neurotransmitter release from nerve terminals. High affinity membrane α‐latrotoxin receptor was purified in an active binding form. It is a membrane glycoprotein (M
r 160,000–220,000) which may be complexed to a smaller polypeptide (M
r 29,000). The structure of the receptor protein suggests that it may be a synapse‐specific cell recognition molecule. Intracellularly, the α‐latrotoxin receptor interacts with synaptotagmin, a calcium‐ and phospholipid‐binding protein specifically localized in the synaptic vesicle membrane. This interaction may be important for targeting of synaptic vesicles to presynaptic release sites.
Identification of the active site serine of hormone‐sensitive lipase by site‐directed mutagenesis
Authors: Holm Cecilia , Davis Richard C. , Østerlund Torben , Schotz Michael C. , Fredrikson Gudrun
Published: July 2002

The consensus pentapeptide GXSXG is found in virtually all lipases/esterases and generally contains the active site serine. The primary sequence of hormone‐sensitive lipase contains a single copy of this pentapeptide, surrounding Ser‐423. We have analyzed the catalytic role of Ser‐423 by site‐directed mutagenesis and expression of the mutant hormone‐sensitive lipase in COS cells. Substitution of Ser‐423 by several different amino acids resulted in the complete abolition of both lipase and esterase activity, whereas mutation of other conserved serine residues had no effect on the catalytic activity. These results strongly suggest that Ser‐423 is the active site serine of hormone‐sensitive lipase.
Inhibition of cellular response to platelet‐derived growth factor by low Mr phosphotyrosine protein phosphatase overexpression
Authors: Berti Andrea , Rigacci Stefania , Raugei Giovanni , Degl'Innocenti Donatella , Ramponi Giampietro
Published: July 2002

The role of low M
r phosphotyrosine protein phosphatase (PTPase) in the control of cell proliferation was studied. A synthetic gene coding for PTPase was transfected and expressed in NIH/3T3 fibroblasts. The effects of the enzyme were particularly evident when cells were stimulated by platelet‐derived growth factor (PDGF). The mitogenic response to PDGF was decreased and the inhibition reached 90%. This effect was more pronounced with respect to fetal calf serum stimulation. Hormone‐dependent autophosphorylation of the PDGF receptor was significantly reduced. These results demonstrate that low M
r PTPase, a cytosolic enzyme, not only affects cellular response to PDGF but also reduces the membrane receptor autophosphorylation.
Protein‐associated pigments that accumulate in the brunescent eye lens
Authors: Luthra Manni , Ranganathan Darshan , Ranganathan Subramania , Balasubramanian Dorairajan
Published: July 2002

Brunescent (dark brown) cataract is particularly prevalent in the tropics. Enzymatic digestion of the insoluble protein fraction of brunescent cataractous eye lenses from India, followed by high performance liquid chromatographic separation of the pigments and spectroscopic investigations, have led to the identification of one of the pigments as 4‐hydroxyquinoline‐3‐[α‐aminoacetic acid] (compound A). The 4‐hydroxyquinoline moiety is shown to be a photodynamic agent that generates O•−
2 and leads to protein crosslinking. This suggests that the compound A may play a long‐term deleterious role in situ in the lens.
Degradation of the yeast MATα2 transcriptional regulator is mediated by the proteasome
Authors: Richter-Ruoff Birgit , Wolf Dieter H. , Hochstrasser Mark
Published: July 2002

Rapid degradation of specific regulatory proteins plays a role in a wide range of cellular phenomena, including cell cycle progression and the regulation of cell growth and differentiation. A major mechanism of selective protein turnover in vivo involves a large multi‐subunit protease known as the proteasome or multi‐catalytic proteinase. At the same time, the degradation of many cellular proteins requires their covalent ligation to the polypeptide ubiquitin. Here we show that the yeast S cerevisiae MATα2 repressor, which is known to be ubiquitinylated in vivo, requires the proteasome for its rapid intracellular proteolysis.
L protein, encoded by psbL, restores normal functioning of the primary quinone acceptor, QA, in isolated D1/D2/CP47/Cytb‐559/I photosystem II reaction center core complex
Authors: Kitamura Kohji , Ozawa Shinichiro , Shiina Takashi , Toyoshima Yoshinori
Published: July 2002

Plastoquinone‐9(PQ‐9)‐depleted PSII reaction center core complex, consisting of CP47/D1/D2/Cytb‐55911, was isolated from spinach PSII particles. PQ‐9, lipids and several proteins were extracted from the original PSII particles and separated by several steps of chromatography to be reconstituted into the isolated complex. PQ‐9 reconstituted in the complex with the help of thylakoid lipids (digalactosyldiglyceride) did not function as QA by itself. However, PQ‐9 simultaneously reconstituted with L protein and the thylakoid lipids successfully functioned as QA in the complex. Other proteins of PSII origin, such as CP43, H, K, nuclear encoded 4.1 and 5.0 kDa proteins, are unable to restore the QA activity in the complex.
Tissue‐dependent developmental expression of a cytosolic thyroid hormone protein gene in Xenopus: Its role in the regulation of amphibian metamorphosis
Authors: Shi Yun-Bo , Liang Vivia C.T. , Parkison Clifford , Cheng Sheue-Yann
Published: July 2002

We have cloned the cDNA encoding the Xenopus laevis homolog of mammalian cytosolic thyroid hormone binding protein (CTHBP). We found that while its mRNA level varies little in whole animals during development, the expression of CTHBP is inversely correlated with tissue‐specific transformations during metamorphosis. A high level of its mRNA was observed in the tail of premetamorphic tadpoles. However, the expression is dramatically repressed with the onset of rapid tail resorption. In the hindlimb, the expression of CTHBP is very low during morphogenesis. Subsequently, its expression continuously increases during the period of limb growth. In contrast, a low level of CTHBP expression was detected in the intestine throughout metamorphosis. These results suggest that CTHBP could function to modulate the metamorphic process by regulating the level of intracellular thyroid hormones.
Thiol ester role in correct folding and conformation of human α2‐macroglobulin
Authors: Gettins Peter G.W. , Boel Esper , Crews Brenda C.
Published: July 2002

To determine the role of the thiol ester in the folding of human α2‐macroglobulin (α2M) in the active conformation, we have characterized a recombinant variant of α2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester‐forming cysteine. C949S α2M behaves like methylamine‐treated plasma α2M, with correctly formed inter‐subunit disulfide bridges, non‐covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric α2M does not require a pre‐formed thiol ester. Active α2M may form in vivo by a two‐step process involving initial folding to give a structure resembling that of C949S α2M followed by thiol ester formation and a conformational change that gives the native active state.
The ATP‐driven Na+‐pump in the plasma membrane of the marine unicellular alga, Platymonas viridis
Authors: Balnokin Yurii V. , Popova Larisa G.
Published: July 2002

The ATP‐supported 22NA+ uptake by plasma membrane vesicles from the marine microalga, Platymonas viridis, was studied. At pH 7 in the medium, Na+ uptake did not occur in the presence of ATP although ΔpH across the plasma membrane was generated. The ATP‐dependent Na+ uptake was induced by adding the protonophore, CICCP. At pH 8, Na+ uptake took place when ATP was added even without CICCP. The ΔpH generated across the plasma membrane was negligible under these conditions. The Na+ uptake at pH 8 was not affected by CICCP and amiloride, an inhibitor of the Na+/H+ antiporter. It is concluded that the ATP‐supported Na+ uptake by Pl. viridis vesicles is catalyzed by Na+‐ATPase.
Automation of micro‐preparation and enzymatic cleavage of gel electrophoretically separated proteins
Authors: Houthaeve Tony , Gausepohl Heinrich , Mann Matthias , Ashman Keith
Published: July 2002

To achieve high throughput, protein microcharacterization sample preparation must be automated. We describe a cartesian robot capable of processing 32 protein samples in parallel. The system is based on specially designed flow‐through reactors for contamination‐free reagent delivery and removal. Washing of excised gel pieces, reduction and alkylation, proteolytic cleavage and peptide extraction are performed in these reactors. Compatibility of the system with HPLC peptide separation and Edman degradation as well as with laser desorption mass spectrometry of the unseparated mixture is demonstrated. This is the first report describing automated preparation and processing of multiple protein samples.
Multidrug‐resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses
Authors: Zini Nicoletta , Scotlandi Katia , Baldini Nicola , Nini Giuseppe , Sabatelli Patrixia , Maraldi Nadir M
Published: July 2002

Summary— Multidrug‐resistant (MDR) variants of a human osteosarcoma cell line (U‐2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P‐glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.
Gastrin‐cholecystokinin‐like immunoreactivity in the central nervous system of the milkweed bug, Spilostethus pandurus. Ultrastructural aspects of the reactive A cells of the brain
Authors: Garcerá Maria Dolores , Tamarelle Mireille
Published: July 2002

Summary— All the ganglia belonging to the central nervous system of adults of the milkweed bug Spilostethus pandurus (Hemiptera) were screened immunohistochemically for vertebrate gastrin‐cholecystokinin (CCK‐8(s))‐like peptides. Several large reactive perikarya are present in the median part of the protocerebrum, their processes extending to the dorsal ‘aorta’. These cell bodies are also paraldehyde fuchsin‐positive, ie they are A‐type cells. In the lateral part of the protocerebrum, in the deutocerebrum and tritocerebrum, and in the suboesophageal, prothoracic and abdominal ganglia, a few immunoreactive cell bodies send axonal processes into their respective neuropiles. The A‐type cells reactive to CCK antiserum were identified, at the ultrastructural level, by combining paraldehyde fuschin staining of semithin sections with a post‐embedding immunogold technique carried out on adjacent ultrathin sections. The neurosecretory cells contain numerous vesicles of elevated electron density. These data suggest that members of the CCK peptide family are present in the central nervous system of Spilostethus pandurus.
Schizosaccharomyces pombe Rad9 contains a BH3‐like region and interacts with the anti‐apoptotic protein Bcl‐2
Authors: Komatsu Kiyoshi , Hopkins Kevin M. , Lieberman Howard B. , Wang Hong-Gang
Published: July 2002

Here we report that the Schizosaccharomyces pombe Rad9 (SpRad9) protein contains a group of amino acids with similarity to the Bcl‐2 homology 3 death domain, which is required for SpRad9 interaction with human Bcl‐2 and apoptosis induction in human cells. Overexpression of Bcl‐2 in S. pombe inhibits cell growth independently of rad9, but enhances resistance of rad9‐null cells to methyl methanesulfonate, ultraviolet and ionizing radiation. These observations suggest that SpRad9 may represent the first member of the Bcl‐2 protein family identified in yeast, though the cell death pathways in S. pombe may differ from those found in mammals.
Ectopic expression of TATA box‐binding protein induces shoot proliferation in Arabidopsis
Authors: Li You-Fang , Dubois Frédéric , Zhou Dao-Xiu
Published: July 2002

The TATA box‐binding protein (TBP) is an essential component of transcription initiation complexes of all three eukaryotic RNA polymerases. Increasing evidence suggests that the TBP activity and availability may be regulated to precisely control gene transcription and play an important role in cell type‐specific regulation. Arabidopsis TBP‐2 is up‐regulated in apical shoot tissues. Overexpression of TBP‐2 in transgenic Arabidopsis induces apical shoot proliferation. The expression of some shoot meristem regulatory genes is altered. These data suggest that the TBP gene dosage and/or expression level may play an important role in controlling shoot production and plant morphology.
N‐terminal PDZ domain is required for NHERF dimerization
Authors: Shenolikar Shirish , Minkoff Charles M. , Steplock Deborah A. , Evangelista Christine , Liu Min-Zhi , Weinman Edward J.
Published: July 2002

NHERF, a 55 kDa PDZ‐containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time‐ and concentration‐dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N‐terminal PDZ‐I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.
Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli
Authors: Li Xiao-Dan , Villa Anthony , Gownley Colleen , Kim Myong Jin , Song Jinmei , Auer Manfred , Wang Da-Neng
Published: July 2002

The γ‐aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His‐ and myc‐tags, and size‐exclusion chromatography. The purified transporter exists as a monomer in FOS‐Choline 12 detergent, with a Stokes radius of 45 Å for the protein–detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K
d) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 μM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.
Intermolecular hydrogen bonding between carotenoid and bacteriochlorophyll in LH2
Authors: He Zhi , Sundström Villy , Pullerits Tõnu
Published: July 2002

We have studied van der Waals contacts of the carotenoid rhodopin glucoside (RG) with the bacteriochlorophyll pigments absorbing at 800 nm (B800) in the crystal structure of Rhodopseudomonas acidophila, and the hydrogen positions were determined from quantum chemical calculations at the Hartree–Fock (6‐31G) level. We have found strong evidence for hydrogen bonding between the B800 BChl and the RG from neighboring protomer units. The binding energy was estimated to be about 2 kcal/mol (700 cm−1). CI‐singles approach and time‐dependent density functional theory calculations of the B800–RG dimer indicate a red‐shift (ca 2 nm) of the B800 Q
y transition, along with a substantial increase of its oscillator strength, probably due to the hydrogen bonding.
pH dependence of the hydrogen exchange in the SH3 domain of α‐spectrin
Authors: Sadqi M. , Casares S. , López-Mayorga O. , Martı́nez J.C. , Conejero-Lara F.
Published: July 2002

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of α‐spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.
Probing phosphatidylinositolphosphates and adenosinenucleotides on talin nucleated actin polymerization
Authors: Isenberg G. , Niggli V. , Pieper U. , Kaufmann S. , Goldmann W.H.
Published: July 2002

We have investigated the binding of PI, PIP and PIP2 to talin and the effect of phosphoinositides and adenosinenucleotides on talin‐induced actin polymerization. At physiological salt concentrations, talin coprecipitates with liposomes when containing phosphoinositides but not when containing PI. The nucleating effect of talin as reflected by a twofold increase of fluorescence during the polymerization of actin labelled with NBD is not inhibited by phosphoinositides. The polymerization of ADP‐actin versus ATP‐actin was investigated in the presence and absence of talin by NBD fluorescence. ADP‐actin nucleation induced by talin is comparably efficient as with ATP‐actin. These experimental findings in summary have implications when evaluating the role of talin during cell activation.
The MHC‐encoded TAP1/LMP2 bidirectional promoter is down‐regulated in highly oncogenic adenovirus type 12 transformed cells
Authors: Proffitt James A , Blair G.Eric
Published: July 2002

Cells transformed by human adenovirus 12 (Ad12) exhibit extremely low surface levels of MHC class I molecules and contain reduced levels of class I heavy chain mRNAs. We report that levels of MHC‐encoded TAP1 and LMP2 mRNAs are also down‐regulated in Ad12‐transformed rat cells, and that transcription of rat TAP1 and LMP2 transcripts is directed from a 564 bp intergenic region which is significantly less active in Ad12‐transformed cells compared to those transformed with Ad5. Our results suggest that, in common with MHC class I gene expression, TAP1 and LMP2 gene expression is reduced mainly at the level of transcription in Ad12‐transformed cells.
The glutamine ligand in the ferrous iron active site of isopenicillin N synthase of Streptomyces jumonjinensis is not essential for catalysis
Authors: Landman Orna , Borovok Ilya , Aharonowitz Yair , Cohen Gerald
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Peroxynitrite oxidises catechols to o‐quinones
Authors: Kerry Nicole , Rice-Evans Catherine
Published: July 2002

Nitration of phenolic compounds is a well‐established mechanism on interaction with peroxynitrite. However, while nitration is the predominant reaction for monophenolic hydroxycinnamates, this does not take place with the catechol‐containing hydroxycinnamate, caffeic acid. The aim of the present study was to investigate the mechanism of the chemical interaction of caffeic acid with peroxynitrite and to characterise the products formed. A novel compound was detected and characterised as the o‐quinone of caffeic acid based on its reaction with nucleophilic thiol compounds, glutathione and l‐cysteine. The same novel product was identified following the oxidation of caffeic acid in alkaline solutions confirming the identity of this species as a caffeic acid oxidation product.
Solution structure of the Ras‐binding domain of RGL
Authors: Kigawa Takanori , Endo Makoto , Ito Yutaka , Shirouzu Mikako , Kikuchi Akira , Yokoyama Shigeyuki
Published: July 2002

The RGL protein, a homolog of the Ral GDP dissociation stimulator (RalGDS), has been identified as a downstream effector of Ras. In the present study, the solution structure of the Ras‐binding domain of RGL (RGL‐RBD) was determined by NMR spectroscopy. The overall fold of RGL‐RBD consists of a five‐stranded β‐sheet and two α‐helices, which is the same topology as that of RalGDS‐RBD. The backbone chemical shift perturbation of RGL‐RBD upon interaction with the GTP analog‐bound Ras was also examined. The solution structure of RGL‐RBD, especially around some of the Ras‐interacting residues, is appreciably different from that of RalGDS‐RBD.
12‐Hydroxyeicosatetraenoic acid upregulates P‐selectin‐induced tissue factor activity on monocytes
Authors: Pellegrini Giuliana , Malandra Riccardo , Celi Alessandro , Furie Barbara C. , Furie Bruce , Lorenzet Roberto
Published: July 2002

12‐Hydroxyeicosatetraenoic acid (12‐HETE), a product of the platelet lipoxygenase pathway, amplifies tissue factor expression by P‐selectin‐stimulated monocytes in a time‐ and dose‐dependent fashion. The same effect is observed when monocytes are incubated with Chinese hamster ovary cells transfected with the P‐selectin cDNA. Both 5‐HETE and leukotriene C4 are inactive in this system. Furthermore, the effect is not dependent on non‐specific monocyte adhesion, since monocytes incubated with CHO cells expressing E‐selectin do not express tissue factor, either in the presence or in the absence of 12‐HETE. These results show that 12‐HETE is a cofactor for the expression of tissue factor by monocytes.
Evidence that carnitine palmitoyltransferase I (CPT I) is expressed in microsomes and peroxisomes of rat liver
Authors: Fraser Fiona , Corstorphine Clark G , Price Nigel T , Zammit Victor A
Published: July 2002

Mitochondria, microsomes and peroxisomes all express overt (cytosol‐facing) carnitine palmitoyltransferase activity that is inhibitable by malonyl‐CoA. The overt carnitine palmitoyltransferase activity (CPTo) associated with the different fractions was measured. Mitochondria accounted for 65% of total cellular CPTo activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. In parallel experiments, rat livers were perfused in situ with medium containing dinitrophenyl (DNP)‐etomoxir in order to inhibit quantitatively and label covalently (with DNP‐etomoxiryl‐CoA) the molecular species responsible for CPTo activity in each of the membrane systems under near‐physiological conditions. In all three membrane fractions, a single protein with an identical molecular mass of approximately 88 000 kDa (p88) was labelled after DNP‐etomoxir perfusion of the liver. The abundance of labelled p88 was quantitatively related to the respective specific activities of CPTo in each fraction. On Western blots the same protein was immunoreactive with three anti‐peptide antibodies raised against linear epitopes of the cytosolic N‐ and C‐domains and of the inter‐membrane space loop (L) domain of the mitochondrial enzyme (L‐CPT I). However, the reaction of the microsomal protein with the anti‐N peptide antibody (raised against epitope Val‐14–Lys‐29 of CPT I) was an order of magnitude stronger than expected from either microsomal CPTo activity or its DNP‐etomoxiryl‐CoA labelling. This suggests that the N‐terminal domain of the microsomal protein differs from that in the mitochondrial or peroxisomal protein. This conclusion was confirmed using antibody back‐titration experiments, in which the binding of anti‐N and anti‐C antibodies by mitochondria and microsomes was quantified.
Regulation of Limulus skeletal muscle contraction
Authors: Ritter Oliver , Haase Hannelore , Morano Ingo
Published: July 2002

Skeletal muscle contraction of Limulus polyphemus, the horseshoe crab, seemed to be regulated in a dual manner, namely Ca2+ binding to the troponin complex as well phosphorylation of the myosin light chains (MLC) by a Ca2+/calmodulin‐dependent myosin light chain kinase. We investigated muscle contraction in Limulus skinned fibers in the presence of Ca2+ and of Ca2+/calmodulin to find out which of the two mechanisms prevails in Limulus skeletal muscle contraction. Although skinned fibers revealed high basal MLC mono‐ and biphosphorylation levels (0.48 mol phosphate/mol 31 kDa MLC; 0.52 mol phosphate/mol 21 kDa MLC), the muscle fibers were fully relaxed at pCa 8. Upon Ca2+ or Ca2+/calmodulin activation, the fibers developed force (357±78.7 mN/mm2; 338±69.7 mN/mm2, respectively) while the MLC phosphorylation remained essentially unchanged. We conclude that Ca2+ activation is the dominant regulatory mechanism in Limulus skeletal muscle contraction.
Fourier‐transform infrared studies on azide‐binding to the binuclear center of the Escherichia coli bo‐type ubiquinol oxidase1
Authors: Tsubaki Motonari , Mogi Tatsushi , Hori Hiroshi
Published: July 2002

Azide‐binding to the heme‐copper binuclear center of bo‐type ubiquinol oxidase from Escherichia coli was investigated with Fourier‐transform infrared spectroscopy. Deconvolution analyses of infrared spectra of the azide (14N3)‐inhibited air‐oxidized form showed a major infrared azide antisymmetric stretching band at 2041 cm−1. An additional band developed at 2062.5 cm−1 during a longer incubation. Isotope substitutions with terminally 15N‐labelled azides did not show a splitting of the major band, indicating that the geometry of the bound azide is mainly in a bridging configuration between high‐spin heme o and CuB. The band at 2062.5 cm−1 showed clear splittings upon substitution with the terminally 15N‐labelled azides, indicating the CuB
2+‐N=N=N structure. Partial reduction of the oxidase with β‐NADH in the presence of azide caused an appearance of new infrared bands at 2038.5 (major) and 2009 (minor) cm−1. The former band also showed clear splittings in the presence of the terminally 15N‐labelled azides, indicating that reduction of low‐spin heme b alters the structure of the binuclear center leading to the Feo
3+‐N=N=N configuration.
Detection of putative Zn(II) binding sites within Escherichia coli RNA polymerase: inconsistency between sequence‐based prediction and 65Zn blotting
Authors: Sujatha Sitaraman , Chatterji Dipankar
Published: July 2002

The availability of repeating ‘Cys’ and/or ‘His’ units in a particular order prompts the prediction of Zn(II) finger motifs in a protein. Escherichia coli RNA polymerase has two tightly bound Zn(II) per molecule of the enzyme as detected by atomic absorption spectroscopy. One Zn(II) was identified to be at the β subunit, whereas the other putative Zn(II) binding site has recently been predicted to be at the N‐terminal half of the β′ subunit, from primary sequence analysis. We show here that the β′ subunit has no ability to bind 65Zn(II). On the other hand, the N‐terminal domain of the α subunit has strong Zn(II) binding ability with no obvious functional implications.
Detection of a novel plasma serine protease during purification of vitamin K‐dependent coagulation factors
Authors: Hunfeld A , Etscheid M , König H , Seitz R , Dodt J
Published: July 2002

A novel serine protease (PHBSP) was purified from human plasma by two chromatographic steps with a final yield of 1.6 mg/l plasma. The protease consists of two disulfide‐bridged chains of about 50 and 30 kDa with the light chain containing the active site of the enzyme. NH2‐terminal sequence analysis revealed identity to the deduced amino acid sequence of HGFA‐like mRNA. The activity of PHBSP is strongly dependent on Ca2+ ions and is efficiently inhibited by α2‐antiplasmin and aprotinin. Possible functions of PHBSP in the hemostatic system are discussed.
Antibodies against Pex14p block ATP‐independent binding of matrix proteins to peroxisomes in vitro
Authors: Lopez-Huertas Eduardo , Oh Jaesung , Baker Alison
Published: July 2002

The membrane protein Pex14p is a key component of the protein import machinery of peroxisomes. Antibodies raised against human Pex14p recognise a 66 kDa protein in sunflower glyoxysomes (HaPex14p) and immunoprecipitate in vitro‐translated Arabidopsis Pex14p (AtPex14p). These antibodies inhibit the ATP‐independent binding to sunflower peroxisome membranes of peroxisome targeting signal type (PTS) 1‐ and PTS2‐targeted matrix proteins, but not an integral membrane protein. These results suggest that Pex14p functions before the ATP‐dependent step of peroxisome assembly.
Loss of heterozygosity in tumor cells requires re‐evaluation: the data are biased by the size‐dependent differential sensitivity of allele detection
Authors: Liu Jian , Zabarovska Veronika I. , Braga Eleonora , Alimov Andrei , Klein George , Zabarovsky Eugene R.
Published: July 2002

Normal tissue contamination of tumors may eclipse the detection of loss of heterozygosity (LOH) by microsatellite analysis and may also hamper isolation of tumor suppressor genes. To test the potential impact of this problem, we prepared artificial mixtures of mouse‐human microcell hybrid lines that carried different alleles of the same chromosome 3 marker. After performing an allele titration assay, we found a consistent difference between the LOH of a high molecular weight (H) allele and the LOH of a low molecular weight (L) allele of the same CA repeat marker. It follows that normal tissue admixtures will be less of a problem when LOH affects a H allele than with a L allele. Random screening of 100 papers published between 1994 and 1999 revealed that the loss of a L allele was recorded at about half the frequency (52%) of loss of a H allele. To avoid this bias, we have developed rules for the evaluation of LOH data. We suggest that the loss of a L allele should be given more weight than the loss of a H allele in LOH studies using microsatellite markers.
Disulfide cross‐linking of subunits F1‐γ and F0I‐PVP(b) results in asymmetric effects on proton translocation in the mitochondrial ATP synthase
Authors: Gaballo Antonio , Zanotti Franco , Raho Gabriella , Papa Sergio
Published: July 2002

A study is presented on the effect of diamide‐induced disulfide cross‐linking of F1‐γ and F0I‐PVP(b) subunits on proton translocation in the mitochondrial ATP synthase. The results show that, upon cross‐linking of these subunits, whilst proton translocation from the A side to the B F1 side is markedly accelerated with decoupling of oxidative phosphorylation, proton translocation in the reverse direction, driven by either ATP hydrolysis or a diffusion potential, is unaffected. These observations reveal further peculiarities of the mechanism of energy transfer in the ATP synthase of coupling membranes.
Hydroalkylation of Aryl Alkenes with Organohalides Catalyzed by Molybdenum Oxido Based Lewis Pairs
Authors: Zwettler Niklas , Dupé Antoine , Klokić Sumea , Milinković Angela , Rodić Dado , Walg Simon , Neshchadin Dmytro , Belaj Ferdinand , Mösch‐Zanetti Nadia C.
Published: June 2020

Three molybdenum(VI) dioxido complexes [MoO2(L)2] bearing Schiff base ligands were reacted with B(C6F5)3 to afford the corresponding adducts [MoO{OB(C6F5)3}(L)2], which were fully characterized. They exhibit Frustrated Lewis‐Pairs reactivity when reacting with silanes. Especially, the [MoO{OB(C6F5)3}(L)2] complex with L=2,4‐dimethyl‐6‐((phenylimino)methyl)phenol proved to be active as catalyst for the hydroalkylation of aryl alkenes with organohalides and for the Atom‐Transfer Radical Addition (ATRA) of organohalides to aliphatic alkenes. A series of gem‐dichloride and gem‐dibromide compounds with potential for further derivatization were synthesized from simple alkenes and organohalides, like chloroform or bromoform, using low catalyst loading.

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Differentiating molecular etiologies of Angelman syndrome through facial phenotyping using deep learning
Authors: Gomez Diego A. , Bird Lynne M. , Fleischer Nicole , Abdul‐Rahman Omar A.
Published: June 2020

Angelman syndrome (AS) is caused by several genetic mechanisms that impair the expression of maternally‐inherited UBE3A through deletions, paternal uniparental disomy (UPD), UBE3A pathogenic variants, or imprinting defects. Current methods of differentiating the etiology require molecular testing, which is sometimes difficult to obtain. Recently, computer‐based facial analysis systems have been used to assist in identifying genetic conditions based on facial phenotypes. We sought to understand if the facial‐recognition system DeepGestalt could find differences in phenotype between molecular subtypes of AS. Images and molecular data on 261 individuals with AS ranging from 10 months through 32 years were analyzed by DeepGestalt in a cross‐validation model with receiver operating characteristic (ROC) curves generated. The area under the curve (AUC) of the ROC for each molecular subtype was compared and ranked from least to greatest differentiable phenotype. We determined that DeepGestalt demonstrated a high degree of discrimination between the deletion subtype and UPD or imprinting defects, and a lower degree of discrimination with the UBE3A pathogenic variants subtype. Our findings suggest that DeepGestalt can recognize subclinical differences in phenotype based on etiology and may provide decision support for testing.
Scale‐up of the cross‐flow flat‐plate airlift photobioreactor
Authors: Zhao Luhaibo , Gu Yu , Peng Ci , Tang Zhiyong
Published: June 2020

To commercialize large‐scale and high‐density microalgae cultivation, a 10L cross‐flow flat‐plate airlift photobioreactor (CFA‐PBR) was designed and amplified to two types of 40L CFA‐PBRs via different scaling‐up methods. The multiphase flow characteristics and bioreaction process of these CFA‐PBRs were studied by experiments and computational fluid dynamics (CFD) simulation. The results show that the parameters of CFA‐PBRs, such as bubble size, gas holdup, mass transfer, and photosynthetic reaction efficiency, mainly conform to the principle of geometric similarity under the same light thickness and superficial gas velocity, which is significant for large‐scale industrial applications of the efficient high‐density algae cultivation.
Impact of model selection on predicted contaminant degradation in water treatment
Authors: Gorzalski Alexander S. , Harrington Gregory W. , Coronell Orlando
Published: June 2020

Modeling contaminant degradation is fundamental to the design, operation, and regulation of reactive contaminants in water treatment reactors. The plug flow reactor (PFR) t10 model has represented residence time distribution (RTD) in pathogen regulation for more than 25 years despite known limitations. Alternative RTD models have been developed, including the tanks‐in‐series (TIS), segregated flow (SF), and reactor network (RN) models. Tracer data were gathered from 35 reactors and their RTDs modeled using the PFR, PFR t10, TIS, SF, and RN models. Predicted contaminant degradation across reactor models was studied, and guidance was developed for selecting RTD models at different log reduction values (LRVs). This study demonstrated that the PFR t10 model may overpredict contaminant degradation at LRVs >2‐log and that the TIS, SF, or RN model should be used for these higher LRVs. A graphical method was also developed for rapidly screening predicted treatment efficacy for a range of contaminants.
A facile synthesis of multifunctional carbon dots as fluorescence ‘turn on’ and ‘turn off’ probes for selective detection of Al3+ and 2,4,6‐trinitrophenol
Authors: Zhao Feifei , Zhang Tianyi , Yang Yu , Lü Changli
Published: June 2020

Carbon dots (CDs) have drawn increasing interests due to their unique optical properties and promising application in various fields. In this study, citric acid (CA) and 5‐chloromethyl‐8‐hydroxyquinoline (LQ) were used to synthesize nitrogen‐doped CDs as novel fluorescent probes using a one‐step solvothermal route. The as‐prepared CDs had strong blue‐white fluorescence emission when excited at 405 nm wavelength with a quantum yield (QY) of 25%, behaving with high ion concentration stability. Water‐soluble CDs with a 8‐hydroxyquinoline structure on their surface could be used to detect Al3+ using a ‘turn on’ mechanism and trinitrophenol (TNP) using a ‘turn off’ mechanism with detection limits of 229 nM and 44.4 nM, respectively. Al3+ enhances the fluorescence of CDs by forming a coordination complex to generate a fluorescence synergistic role and limit CD nonradiative transition. TNP quenched the fluorescence with high selectivity and sensitivity, which was attributed to the inner filter effect and static quenching. These results indicated that these CDs with their unique ‘turn on’ and ‘turn off’ nature have potential application in the environmental protection field and in prevention of terrorist threats.