bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression, purification and crystallization of human phosphotyrosine phosphatase 1B
Authors: HOPPE Edmund , BERNE Pierre‐François , STOCK Daniela , RASMUSSEN Jesper Skou , MØLLER Niels Peter H. , ULLRICH Axel , HUBER Robert
Published: March 2005

Protein phosphotyrosine phosphatases are believed to be involved in the regulation of the activity of cellular proteins, such as receptor tyrosine kinases, by controlling their phosphorylation status. One of the best described and characterized protein of this class of enzymes is the phosphotyrosine phosphatase 1B. To obtain sufficient quantities for structural investigations, truncated forms of PTP1B encompassing the catalytic domain were over‐expressed in Escherichia coli and purified to apparent homogeneity by conventional chromatography. The activity of these purified enzymes has been compared with the wild‐type enzyme expressed in mammalian cells. By measuring the activities against p‐nitrophenyl phosphate, the pH dependence of this activity, and responses to different modulators, it could be demonstrated that the truncated forms of PTP1B retained the same characteristics as the full‐length mammalian enzyme, but are not subject to inhibition of enzymic activity mediated by the C‐terminus. Due to their improved solubility, it can be assumed that the catalytic domains are advantageous for crystallization studies in comparison to the natural enzyme. In a screening for crystallization conditions, we obtained protein crystals indicating that the quality of the purified protein is sufficient for crystallographic studies.
Solution structure of the variant‐3 neurotoxin from Centruroides sculpturatus Ewing
Authors: LEE Weontae , MOORE Carolynn H. , WATT Dean D. , KRISHNA N. Rama
Published: March 2005

The solution structure of the CsE‐v3 neurotoxin from the venom of the North American scorpion Centruroides sculpturatus Ewing (CsE) has been determined by a hybrid refinement procedure that employed distance geometry and dynamical simulated annealing. Distance constraints deduced from the nuclear Overhauser effect spectroscopy data and torsion angle constraints deduced from the vicinal coupling constant data were used in the refinement procedure. A family of simulated annealing structures that showed no constraint violations was generated. The energy‐minimized average structure exhibited root‐mean‐square deviations of 0.121 nm for the backbone and 0.182 nm for all atoms, with respect to this family. These studies confirm the previously qualitative NMR findings about the secondary structural features, viz. the presence of a short α‐helix composed of residues 23–31 and an antiparallel β‐sheet composed of the strands of residues 1–5, 45–50 and 36–42. A cluster of aromatic ring systems is located on one side of the protein. The solution and crystal structures have similar overall features, but show some minor differences.
Isolation and structure of novel defensive peptides from frog skin
Authors: MOR Amram , NICOLAS Pierre
Published: March 2005

In addition to the highly specific cell‐mediated immune system, vertebrates possess an efficient host‐defense mechanism against invading microorganisms which involves the synthesis of highly potent antimicrobial peptides with a large spectrum of activity. A 34‐residue cationic and amphiphatic peptide, designated dermaseptin I, was recently isolated from the skin of the arboreal frog Phyllomedusa sauvagii and was shown to exhibit microbicidal activity against various pathogenic microorganisms including bacteria, yeast, protozoa and filamentous fungi. In this study, we report the isolation and characterization of four novel antimicrobial peptides from frog skin through the combined use of an anti‐dermaseptin enzyme immunoassay and an antifungal bioassay. The 28‐34‐residue antimicrobial peptides are cationic, containing 3–5 lysine residues that punctuate an alternating hydrophobic and hydrophilic sequence. Based on their primary structure, all four peptides can be fitted to a class L amphipathic α helix which places all lysine residues on the polar side of the helix. The four antimicrobial peptides have high sequence similarity with dermaseptin I (53–94% similarity) suggesting that their respective genes are all members of the same family. In addition, pairwise sequence alignment of dermaseptin I and adenoregulin, a 33‐residue cationic peptide recently isolated from frog skin and shown to enhance the binding of agonists to the A1 adenosine receptor, reveals 62% similarity (39% amino acid positional identity). Both peptides share a similar but non‐identical spectrum of antimicrobial activity, being active against bacteria, yeast and filamentous molds. However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic cells. These findings indicate that adenoregulin should be included in the dermaseptin family of peptides. In addition, tryptic digestion of purified pro‐dermaseptin I liberated a 15‐amino‐acid peptide identified as the authentic C‐terminus of dermaseptin I. These results are in accordance with the predicted sequences of pro‐dermaseptins obtained through molecular cloning, in which the dermaseptin progenitor sequences are located at the extreme C‐terminus of the precursors.
Reversible conformational transition gives rise to ‘zig‐zag’ temperature dependence of the rate constant of irreversible thermoinactivation of enzymes
Authors: LEVITSKY Vladislav Yu. , MELIK‐NUBAROV Nikolay S. , SIKSNIS Virginius A. , GRINBERG Valeriy Ya. , BUROVA Tatyana V. , LEVASHOV Andrey V. , MOZHAEV Vadim V.
Published: March 2005

We have obtained unusual ‘zig‐zag’ temperature dependencies of the rate constant of irreversible thermoinactivation (kin) of enzymes (α‐chymotrypsin, covalently modified α‐chymotrypsin, and ribonuclease) in a plot of log kin versus reciprocal temperature (Arrhenius plot). These dependencies are characterized by the presence of both ascending and descending linear portions which have positive and negative values of the effective activation energy (Ea), respectively. A kinetic scheme has been suggested that fits best for a description of these zig‐zag dependencies. A key element of this scheme is the temperature‐dependent reversible conformational transition of enzyme from the ‘low‐temperature’ native state to a ‘high‐temperature’ denatured form; the latter form is significantly more stable against irreversible thermoinactivation than the native enzyme. A possible explanation for a difference in thermal stabilities is that low‐temperature and high‐temperature forms are inactivated according to different mechanisms. Existence of the suggested conformational transition was proved by the methods of fluorescence spectroscopy and differential scanning calorimetry. The values of ΔH and ΔS for this transition, determined from calorimetric experiments, are highly positive; this fact underlies a conclusion that this heat‐induced transition is caused by an unfolding of the protein molecule. Surprisingly, in the unfolded high‐temperature conformation, α‐chymotrypsin has a pronounced proteolytic activity, although this activity is much smaller than that of the native enzyme.
Chlorophyll synthetase cannot synthesize chlorophyll a′
Authors: HELFRICH Michael , SCHOCH Siegrid , LEMPERT Ulrika , CMIEL Edmund , RÜDIGER Wolfhart
Published: March 2005

Chlorophyll synthetase catalyzes the last step of chlorophyll biosynthesis, namely prenylation (esterification) of chlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate. During investigation of various chlorophyllide derivatives as potential substrates we observed lower esterification with increasing percentages of chlorophyllide a′ in epimeric mixtures of chlorophyllides a and a′. To avoid epimerization during esterification, we studied the reaction in detail with model compounds [zinc‐132(R)‐methoxy‐pheophorbide a and zinc‐132(S)‐methoxy‐pheophorbide a, zinc‐132(R)‐methoxy‐pyropheophorbide a and zinc‐chlorin e6‐131, 152‐dimethylester]. We conclude that compounds which have the 132‐carbomethoxy group at the same side of the macrocycle as the propionic side chain of ring D are neither substrates nor competitive inhibitors. Only compounds having the 132‐carbomethoxy group at the opposite site are substrates for the enzyme. Naturally occuring chlorophyll a′ must be formed by epimerization after esterification.
Leukotriene B4 formation upon halothane‐induced lipid peroxidation in liver membrane fractions under low O2 concentrations in vitro
Authors: GUT Josef , HUWYLER Jörg
Published: March 2005

Lipid peroxidation was induced in rat liver membrane fractions in vitro upon NADPH‐dependent metabolic activation of the anesthetic agent halothane at low O2 concentrations. Halothane‐induced lipid peroxidation was dependent on time, concentration of halothane, and the calculated O2 concentrations present in the system. Lipid peroxidation was inducible at increasing O2 concentrations up to 12 μM, decreased at higher O2 concentrations up to 48 μM, and was not detectable at normoxic conditions. Leukotriene B4 (LTB4) was identified as a product arising upon lipid peroxidation by reverse‐phase high‐pressure liquid chromatography combined with a radioimmunoassay. LTB4 formation was maximal under conditions of maximal lipid peroxidation at a calculated O2 concentration of 12 μM. Even at high concentrations, the 5‐lipoxygenase inhibitors MK886 (10 μM), ZD2138 (20 μM), and ZM230487 (20 μM) were not inhibitory in halothane‐induced lipid peroxidation nor in the associated formation of LTB4. Synthetic LTB4 was transformed into its 20‐hydroxy derivative by ω‐oxidation in an O2‐concentration‐dependent manner, being considerably reduced at the low O2 concentrations that maximally promoted lipid peroxidation. The collective evidence of these data raises the possibility that exposure to halothane might lead to peroxidation‐associated net synthesis of LTB4 through 5‐lipoxygenase‐independent escape routes in liver tissue under physiologically or pathophysiologically low O2 concentrations.
Cloning of human acetyl‐CoA carboxylase cDNA
Authors: HA Joohun , DANIEL Samira , KONG In‐Soo , PARK Chung‐Kil , TAE Hyi‐Jeong , KIM Ki‐Han
Published: March 2005

Acetyl‐CoA carboxylase is the rate‐limiting enzyme in the biogenesis of long‐chain fatty acids. In order to understand the mechanisms that regulate human acetyl‐CoA carboxylase at the gene level, and the relationship between its structure and function, cDNA clones for human acetyl‐CoA carboxylase have been isolated and sequenced. Human acetyl‐CoA‐carboxylase cDNA contains 7020 nucleotides encoding a protein of 2340 amino acids with a calculated relative molecular mass of 264575. The human enzyme shows approximately 85% identity in nucleotide sequence with previously cloned rat acetyl‐CoA carboxylase, and shows 90% identity in the amino acid sequence. Two human acetyl‐CoA‐carboxylase mRNA species, which differ in the 5′ untranslated region with the same coding sequence, have been identified. The sequence analysis reveals that type I and type II acetyl‐CoA‐carboxylase mRNA contain 313‐ and 173‐base‐long 5′ untranslated regions, respectively. The first 240 nucleotides in the 5′ untranslated region of type I acetyl‐CoA‐carboxylase mRNA replace the first 100 nucleotides of the (G+C)‐rich region of the 5′ untranslated region of the type II mRNA. These two species of mRNAs are the only species of human ACC mRNA which have been detected compared to at least five species in rat tissues, and they are expressed in a tissue‐specific manner.
The molecular forms of acetylcholinesterase from Necator americanus (Nematoda), a hookworm parasite of the human intestine
Authors: PRITCHARD David I. , BROWN Alan , TOUTANT Jean‐Pierre
Published: March 2005

Necator americanus (Nematoda: Strongyloidea), a human hookworm parasite, is known to release considerable amounts of acetylcholinesterase (AChE) [Pritchard, D. I., Leggett K. V., Rogan, M. T., McKean, P. G. & Brown, A. (1991) Necator americanus secretory acetylcholinesterase and its purification from excretory/secretory products by affinity chromatography, Parasite Immunol. 13, 187–199]. The present study deals with AChE activity recovered in sequential somatic extracts, and excretory/secretory products, of the adult stage of the parasite. 97% of AChE was extractable in low‐salt and high‐salt detergent‐free buffers, and only 3% was solubilised by a further extraction in the presence of Triton X‐100. AChE in all three extracts was affected by the AChE inhibitors eserine, bis(4‐allyldimethylammoniumphenyl)pentan‐3‐one dibromide and edrophonium chloride, but was resistant to the effects of tetramonoisopropylpyrophosphortetramide, a butyrylcholinesterase inhibitor. Sucrose density centrifugation revealed that AChE in all somatic extracts (low‐salt, high‐salt and detergent) resolved almost exclusively as a single peak between 6.9–7.5 S, while excretory/secretory products resolved at 8.2 S. These values are all compatible with dimers of catalytic sub‐units and no evidence was found for the presence of higher oligomers such as asymmetric forms. The only sample to show a shift in sedimentation following the inclusion of detergent (Triton X‐100, Brij 96) in the gradient was a component of the detergent‐soluble extract, indicating the existence of a minor amphiphilic form. In low‐salt‐soluble and high‐salt‐soluble extracts, AChE was solubilised as a hydrophilic globular form, probably a dimeric G2. The analysis of diisopropylfluorophosphate‐labelled extracts by SDS/PAGE, and unlabelled extracts by immunoblotting using a polyvalent antiserum to N. americanus AChE, indicated that the AChE isolated in each extract was biochemically and immunologically similar. The banding patterns obtained were comparable to that seen when purified AChE was analysed by SDS/PAGE and immunoblotted. This suggests that the basic catalytic subunit has a mass of 66–70 kDa with the active site being located in a 30‐kDa domain. All experimental data indicate the existence of only one AChE class in Necator homologous to AChE of class B from Caenorhabditis elegans. The solubility characteristics and globular nature of this hookworm AChE suggest that its major function is as an excretory or secretory product. This again raises the question of the true biological function of this ‘non‐cholinergenic’ nematode secretion.
Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain
Authors: PFEIFFER Günter , STRUBE Karl‐Hermann , SCHMIDT Martin , GEYER Rudolf
Published: March 2005

Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N‐glycosylation site in the epidermal‐growth‐factor‐like domain due to the replacement of either Tyr67 by Asn (YN‐tPA) or Gly60 by Ser (GS‐tPA) were expressed in mouse epithelial cells (C127) in the presence of [6‐3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo‐β‐N‐acetylglucosaminidase H or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high‐pH anion‐exchange chromatography (high‐mannose and hybrid‐type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex‐type sugar chains). Individual (sub)fractions of glycans were studied by methylation analysis, liquid secondary‐ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N‐terminal amino acid sequencing.

The results revealed that Asn117 of YN‐tPA carried exclusively high‐mannose‐type glycans with five to nine mannose residues similar to wild‐type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.‐H. & Geyer, R. (1989) Eur. J. Biochem. 186, 273–286]. In contrast, Asn117 of GS‐tPA carried only small amounts (about 25%) of high‐mannose and hybrid‐type species and predominantly complex‐type sugar chains (about 75%) which were partially incomplete and mostly devoid of fucose. Newly introduced N‐glycosylation sites at Asn67 (YN‐tPA) or Asn58 (GS‐tPA) as well as those at Asn184 and Asn448 were solely substituted by complex‐type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Galα3‐residues, sulphate groups, intersecting GlcNAc and lactosamine repeats.

Our study clearly demonstrates that creation of a new glycosylation site at Asn58 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn117, whereas introduction of a new site at Asn67 did not. The relative amounts of complex‐type glycans at Asn117 of GS‐tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at that position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS‐tPA glycosylation.
Cloning and expression of cDNA of human Δ4‐3‐oxosteroid 5β‐reductase and substrate specificity of the expressed enzyme
Authors: KONDO Kazu‐Hiro , KAI Masa‐Hiro , SETOGUCHI Yoshiko , EGGERTSEN Gösta , SJÖBLOM Peter , SETOGUCHI Toshiaki , OKUDA Kyu‐Ichiro , BJÖRKHEM Ingemar
Published: March 2005

The enzyme Δ4‐3‐oxosteroid 5β‐reductase (3‐oxo‐5β‐steroid: NADP+ oxidoreductase and 4,5β‐dihydrocortisone: NADP+Δ4‐oxidoreductase) catalyzes the reduction of the Δ4 double bond of bile acid intermediates and steroid hormones carrying the Δ4‐3‐one structure in the A/B cis configuration. Human Δ4‐3‐oxosteroid 5β‐reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y., Noshiro, M., Shimosato, T. & Okuda, K.‐I. (1991) FEBS Lett. 283, 215–218]. DNA sequence analysis of a hybridization‐positive clone predicted the human Δ4‐3‐oxosteroid 5β‐reductase to contain 326 amino acids. The amino acid sequence of the human Δ4‐3‐oxosteroid 5β‐reductase had 79% overall identity to the rat enzyme sequence. It also showed 54% and 50% overall identity with rat 3α‐hydroxysteroid dehydrogenase and human aldose reductase, respectively. RNA blotting analysis demonstrated the existence of a single Δ4‐3‐oxosteroid 5β‐reductase mRNA of approximately 2.7 kb in human liver. Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7α,12α‐dihydroxy‐4‐cholesten‐3‐one and 7α‐hydroxy‐4‐cholesten‐3‐one. In addition, the expressed enzyme showed a small but significant 5β‐reduction activity toward 11β,17α,21‐trihydroxy‐Δ4‐pregnene‐3,20‐dione (cortisol) and 17β‐hydroxy‐Δ4‐androsten‐3‐one (testosterone) whereas no activity was observed toward Δ4‐pregnene‐3,20‐dione (progesterone) or Δ4‐androstene‐3‐17‐dione (androstenedione). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones.
Characterisation of a trisulphide derivative of biosynthetic human growth hormone produced in Escherichia coli
Authors: JESPERSEN Anne Munk , CHRISTENSEN Thorkild , KLAUSEN Niels Kristian , NIELSEN Per Franklin , SØRENSEN Hans Holmegaard
Published: March 2005

A novel protein derivative has been found during process development of biosynthetic human growth hormone; it has been characterised as human growth hormone with a Cys182‐Cys189 trisulphide bridge. We have not been able to find a previous report in the literature about this kind of derivative. The characterisation was obtained partly on the full‐length derivative and partly on a tryptic fragment of the derivative. The full‐length derivative was characterised by reduction with 1,4‐dithiothreitol followed by electrospray mass spectrometry, treatment with cysteine and measurement of hydrogen sulphide liberation upon cysteine treatment. The tryptic fragment from peptide mapping was characterised by amino acid analysis, amino acid sequencing and mass spectrometry. All data indicated an extra sulphur atom in the Cys182‐Cys189 cystine bridge.
Molecular cloning and expression of chick embryo Galβ1,4GlcNAcα2,6‐sialyltransferase
Authors: KUROSAWA Nobuyuki , KAWASAKI Mikiko , HAMAMOTO Toshiro , NAKAOKA Takashi , LEE Young‐Choon , ARITA Masanobu , TSUJI Shuichi
Published: March 2005

DNA clones encoding β‐galactoside α2,6‐sialyltransferase have been isolated from chick embryonic cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open‐reading frame coding for 413 amino acids, and the deduced amino acid sequence showed 57.6% identity with the sequence of rat liver Galβ1,4GlcNAcα2,6‐sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, similar to structures found in other glycosyltransferases, consisting of a short N‐terminal cytoplasmic domain, a signal‐membrane anchor domain, a proteolytically sensitive stem region and a large C‐terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the N‐terminus part including the cytoplasmic tail, signal anchor domain and stem region was replaced with an immunoglobulin signal peptide sequence. The expression of this recombinant protein in COS‐7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity only towards the disaccharide moiety of Galβ1,4GlcNAc in glycoproteins.
Purification and characterization of ATBP, a novel protein that binds to A/T stretches in three segments of the Sarcophaga lectin gene
Authors: MATSUI Mayumi , KOBAYASHI Ayako , KUBO Takeo , NATORI Shunji
Published: March 2005

A DNA‐binding protein for the Sarcophaga lectin gene, ATBP (A/T‐stretches‐binding protein) was purified to homogeneity from the nuclear extract of NIH‐Sape‐4 cells. The molecular mass of ATBP determined under denaturing conditions was 53 kDa, but its native molecular mass estimated by gel‐filtration chromatography was 430 kDa, suggesting that it is an octamer of the 53‐kDa subunit. This protein bound to at least three DNA fragments from the Sarcophaga lectin gene, two of them are in the 5′‐upstream region and the other is in a region containing an intron. These fragments are very AT rich and inlaid with stretches of A or T residues (A/T stretches). ATBP was found to have affinity for poly[d(A‐T)]. These results suggest that ATBP binds to A/T stretches in the three DNA fragments from this gene. Furthermore, longer DNA was found to be bound more effectively, suggesting that an octamer of the 53‐kDa subunit has multiple binding sites for the DNA fragments and requires a relatively long DNA sequence for binding.
In vivo incorporation of copper into the iron‐exchangeable and manganese‐exchangable superoxide dismutase from Propionibacterium shermanii
Authors: MEIER Beate , SEHN Anja P. , SCHININÀ M. Eugenia , BARRA Donatella
Published: March 2005

Propionibacterium shermanii, an aerotolerant anaerobe, produces an iron‐containing or a manganese‐containing superoxide dismutase, depending on the metal supplied in the culture medium [Meier, B., Barra, D., Bossa, F., Calabrese, L. & Rotilio, G. (1982) J. Biol. Chem. 257, 13977–13980]. In this study, we demostrate in vivo incorporation of copper into an active superoxide‐dismutase protein when iron and manganese are absent from the growth medium. Superoxide dismutases containing either iron, manganese or copper were isolated from P. shermanii, their complete amino acid sequences were determined and the identity of their protein moieties was established. The polypeptide chain is made up of 201 amino acid residues, corresponding to a molecular mass of 22.6 kDa. From sedimentation equilibrium experiments, the native protein shows a molecular mass of approximately 86 kDa and therefore consists of four identical subunits. The primary structure was compared with the structure of other Fe‐superoxide dismutases and Mn‐superoxide dismutases, in particular those possessing a strict metal cofactor specificity.
Constitutive expression of functional P‐glycoprotein in rat hepatoma cells
Authors: FARDEL Olivier , LOYER Pascal , LECUREUR Valérie , GLAISE Denise , GUILLOUZO André
Published: March 2005

P‐glycoprotein is a plasma‐membrane glycoprotein involved in multidrug resistance. P‐glycoprotein overexpression has been demonstrated to occur in tumor cells after cytotoxic drug exposure, but also in some cancers including hepatocellular carcinomas before any chemotherapeutic treatment. In order to better analyze this constitutive type of tumoral drug resistance, we have investigated P‐glycoprotein expression and function in rat liver tumors induced experimentally by administration of diethylnitrosamine and in two cell clones derived from one of these tumors designated as RHC1 and RHC2. High levels of P‐glycoprotein mRNAs were found in both liver tumor samples and the two hepatoma cell clones as assessed by Northern blotting; both RHC1 and RHC2 cells displayed altered liver functions commonly observed in rat hepatoma cells, particularly the decreased expression of albumin and overexpression of the fetal glutathione S‐transferase 7–7. The use of specific multidrug resistance (mdr) probes revealed a major induction of the mdr1 gene in liver tumor samples while RHC1 and RHC2 cells expressed both mdr1 and mdr3 genes without displaying a major alteration in the number of mdr gene copies as assessed by Southern blotting. High amounts of P‐glycoprotein were also demonstrated in RHC1 and RHC2 cells by Western blotting. These cells were strongly resistant to doxorubicin and vinblastine, two anticancer drugs transported by P‐glycoprotein. Doxorubicin intracellular retention was low in RHC1 and RHC2 cells, but was strongly enhanced in the presence of verapamil, a known modulator agent of P‐glycoprotein; low retention appeared to occur via a drug efflux mechanism, indicating that P‐glycoprotein was fully active. These results show that rat hepatoma cells can display elevated levels of functional P‐glycoprotein without any prior cytotoxic drug selection and suggest that these cells represent a useful model for analyzing P‐glycoprotein regulation in intrinsically clinical drug‐resistant cancers.
Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα
Authors: DIB Karim , EL JAMALI Amina , JACQUEMIN Claude , CORRÈZE Claude
Published: March 2005

The existence of a cAMP‐dependent regulation of the expression of the α‐subunit of the stimulatory guanine nucleotide‐binding protein (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3–6h with isoproterenol (10 μM) or forskolin (10 μM) (a cAMP‐inducing agent) increased (200–400%) the response of adenyllcyclase to agents which bypass the receptor; GTP, GTP[S] or forskolin. For prolonged exposure times (15 h or more) to isoproterenol or forskolin, the adenylylcyclase activity decreased to the value observed in control cells. The same biphasic response of adenylylcyclase to isoproterenol (10 μM) plus GTP (10 μM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished response to isoproterenol was observed in isoproterenol‐treated cells, indicating that the β‐adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the αsubunits of the guanine‐nucleotide binding protein (Gs and Gi) by Western‐blot analysis. The culture of astroglial cells with isoproterenol or forskolin (3–24 h) resulted in a transient increase of both the Gsα and the Giα protein levels, while the level of Gβ subunits was unaffected.

We also identified Gsα protein (about 40% of the total cellular protein) in the supernatant fraction of astroglial cells but its level was not modified by the stimulation of cells by forskolin.

The level of Gsα mRNA measured by Northern‐blot analysis was transiently increased (200%) after stimulation of astroglial cells with isoproterenol or forskolin for an incubation period of 6–9 h, then returned to that of control cells for longer period of time. In addition, the Gsα mRNA level was threefold increased when cells were cultured for 2–6 h with 8‐bromoadenosine 3′:5′‐cyclic monophosphate (10 μM), a permeant analogue of cAMP. These results indicate that cAMP induces a time‐dependent increase of Gsα mRNA.

The half‐life of Gsα protein and Gsα mRNA were determined. Pulse‐chase studies revealed that the decay of Gsα protein was clearly biphasic with an early phase (5–6 h) and a slower second phase (20–25 h) but the treatment of cells with forskolin did not accelerate or slow down the turnover of Gsα protein.

The use of actinomycin D to estimate the stability of Gsα mRNA showed that the half‐life of Gsα mRNA was approximately 7–8 h and was not different in cells treated with 8‐bromoadenosine 3′:5′‐cyclic monophosphate and in non‐treated cells.

In contrast, treatment of cells with cycloheximide led to a significant increase of Gsα mRNA but did not abolish the effect of forskolin.

These studies suggest that the expression of Gsα mRNA is regulated at the transcriptional level and does not require protein synthesis.
Induction of lactase biosynthesis in the human intestinal epithelial cell line Caco‐2
Authors: HAURI Hans‐Peter , SANDER Brigitte , NAIM Hassan
Published: March 2005

The human colonic adenocarcinoma cell line Caco‐2 forms monolayers of differentiated enterocyte‐like cells when cultured on permeable supports. After confluency, Caco‐2 cells express a number of brush‐border enzymes including lactase‐phlorizin hydrolase, sucrase‐isomaltase and dipeptidylpeptidase IV. We have studied, with particular emphasis on lactase‐phlorizin hydrolase, the modulation of biosynthesis of these enzymes by stimulating second messenger systems. Forskolin induced lactase‐phlorizin hydrolase synthesis approximately fourfold within 7 h, suppressed sucraseisomaltase synthesis, and had little effect on dipeptidylpeptidase IV. Dibutyryl‐cAMP. 8‐bromo‐cAMP and vasoactive intestinal peptide also increased lactase‐phlorizin hydrolase biosynthesis, indicating c‐AMP dependent regulation. The induction of lactase‐phlorizin hydrolase biosynthesis could be inhibited by actinomycin D and was preceded by a fourfold increase in lactase‐phlorizin hydrolase mRNA levels, suggesting transcriptional control. Phorbol 12‐myristate 13‐acetate had an inhibitory effect on brush‐border enzyme synthesis, in particular on sucrase‐isomaltase, and blocked the forskolin‐induced biosynthesis of lactase‐phlorizin hydrolase. Lactase‐phlorizin hydrolase synthesis was also inducible by hydrocortisone, but maximal induction required at least 3 days during which time sucrase‐isomaltase synthesis diminished. The results indicate opposite regulation of lactase‐phlorizin hydrolase and sucrase‐isomaltase via cAMP and corticosteroids, and suggest that the Caco‐2 cell line can serve as a model system to study aspects of the humoral regulation of human intestinal brush‐border enzymes in cell culture.
Functional differences in the promoters of the interferon‐inducible (2′‐5′)A oligoadenylate synthetase and 6–16 genes in interferon‐resistant Daudi cells
Authors: GUILLE Matthew J. , LAXTON Carl D. , RUTHERFORD Michael N. , WILLIAMS Bryan R. G. , KERR Ian M.
Published: March 2005

A clone of interferon‐α‐resistant (IFNr) Daudi cells retained much greater transcriptional inducibility of the (2′–5′) oligoadenylate synthetase than the 6–16 gene despite the fact that the response of both genes is mediated by highly similar interferon‐stimulable DNA response elements (ISRE). The primary IFN‐α activatable transcription factor E (ISGF3) and the additional IFN‐α‐inducible ISRE‐binding complex M were greatly reduced in the IFNr cells. The defect in E was in the Eα subunit. In electrophoretic mobility‐shift assays the 6–16 and (2′–5′) oligoadenylate synthetase ISRE competed approximately equivalently for E and M. Moreover although active in wild‐type cells the (2′–5′) oligoadenylate synthetase ISRE was no more capable of conferring inducibility on a reporter gene in the IFNr cells than was the 6–16 ISRE. The contrasting response of the endogenous (2′–5′) oligoadenylate synthetase and 6–16 genes in the IFNr cells is, therefore, unlikely simply to reflect the slight difference in the sequence of their ISRE. Consistent with this, in addition to the ISRE, sequence 5′ to the ISRE in the (2′–5′) oligoadenylate synthetase promoter appeared necessary for good induction by IFNα in the IFNr cells. Subtle quantitative changes in the phenotype of the IFNr cells have, however, precluded a more precise definition of the DNA element(s) involved.
Fluorescence resonance energy transfer within the regulatory light chain of myosin
Authors: BOEY Wylinn , HUANG William , BENNETTS Bruce , SPARROW John , DOS REMEDIOS Cristobal , HAMBLY Brett
Published: March 2005

Rabbit skeletal muscle myosin regulatory light chain‐2 (LC2) contains two reactive cysteine residues, Cys125 and Cys154, and one tryptophan at position 137. Using wild‐type rabbit LC2 or its genetically engineered mutant with Cys125→Arg (C125R), these residues can be selectively modified with fluorescent or chromophoric probes for spectroscopic studies. We have bound suitable donor/acceptor probe pairs to the two cysteine residues and Trp137 in LC2 or C125R, and measured the distance in solution between the probes by fluorescence resonance energy transfer spectroscopy. C125R was made to facilitate specific labelling of the less reactive Cys154, thus allowing the distance between Cys154 and Trp137 to be measured. Our measurements show that these residues are in close proximity to each other, the distance between them ranging from 1.7 nm (between Cys125 and Trp137) to 2.7 nm (Cys125 and Cys154). These results suggest that Cys125, Trp137 and Cys154, spanning up to 29 residues in the sequence of LC2, are spatially close, consistent with these residues residing within a C‐terminal globular domain. The distances we obtained are in agreement with previous crosslinking studies [Huber, P. A., Brunner, U. T. & Schaub, M. C. (1989) Biochemistry 28, 9116–9123; Saraswat, L. & Lowey, S. (1991) J. Biol. Chem. 266, 19777–19785] and structure predictions of LC2. LC2 is located at the head–rod junction of the myosin crossbridge, and provides the primary regulatory mechanism in molluscan and smooth muscle. In skeletal muscle, its functional role is unclear, although it has been implicated in modulating actomyosin interaction [Metzger, J. M. & Moss, R. L. (1992) Biophys. J. 63, 460–468]. The incorporation of spectroscopic probes onto the light chains of myosin in solution or in fibres has become a valuable tool for evaluating the dynamic properties of the crossbridge during force generation.
The amino acid sequence of iguana (Iguana iguana) pancreatic ribonuclease
Authors: ZHAO Wei , BEINTEMA Jaap J. , HOFSTEENGE Jan
Published: March 2005

The pyrimidine‐specific ribonuclease superfamily constitutes a group of homologous proteins so far found only in higher vertebrates. Four separate families are found in mammals, which have resulted from gene duplications in mammalian ancestors. To learn more about the evolutionary history of this superfamily, the primary structure and other characteristics of the pancreatic enzyme from iguana (Iguana iguana), a herbivorous lizard species belonging to the reptiles, have been determined. The polypeptide chain consists of 119 amino acid residues. The positions of insertions and deletions in the sequence are identical to those in the enzyme from snapping turtle. However, the two enzymes differ at 54% of the amino acid positions. Iguana ribonuclease contains no carbohydrate, although the enzyme possesses three recognition sites for carbohydrate attachment, and has a high number of acidic residues in a localized part of the sequence.
1H one‐dimensional and two‐dimensional NMR studies of the ferricytochrome c 551 from Rhodocyclus gelatinosus
Authors: BANCI Lucia , BERTINI Ivano , CAMBRIA Maria Teresa , CAPOZZI Francesco , DIKIY Alexander
Published: March 2005

1H two‐dimensional NMR spectroscopy has been applied to the oxidized form of cytochrome c 551 from Rhodocyclus gelatinosus, which is paramagnetic with S= 1/2. The investigation has allowed a complete and unambiguous assignment of the heme protons and some residues around the heme. We have learned that: the conformation of the axial methionine is equal to that of horse heart cytochrome c and different from two isoenzymes of the same cytochrome c 551 from a different strain; pKa of 6.6±0.3 has been detected through the shift variations of seventh propionate protons. The detailed differences with other cytochromes c in the hyperfine shifts are discussed.
Secondary structure and signal assignments of human‐immunodeficiency‐virus‐1 protease complexed to a novel, structure‐based inhibitor
Authors: YAMAZAKI Toshimasa , NICHOLSON Linda K. , TORCHIA Dennis A. , STAHL Stephan J. , KAUFMAN Joshua D. , WINGFIELD Paul T. , DOMAILLE Peter J. , CAMPBELL‐BURK Sharon
Published: March 2005

We report comprehensive NMR studies in solution of the human‐immunodeficiency‐virus (HIV)‐1 protease. Stable solutions of the protease were obtained by complexing the protein to a designed cyclic urea inhibitor DMP 323. A variety of triple‐resonance experiments provided essentially complete 1H, 13C and 15N NMR signal assignments of the protease. These assignments, together with short‐range NOE constraints, coupling constants and hydrogen‐exchange data, yielded the secondary structure of the protease in solution. The results reported herein open the way to the determination of the high‐resolution three‐dimensional solution structures of protease/inhibitor complexes, as well as to studies of protease dynamics and solvent interactions.
Expression, Cytoskeletal Utilization and Dimer Formation of Tropomyosin Derived from Retroviral‐Mediated cDNA Transfer
Authors: Prasad Gaddamanugu L. , Fuldner Rebecca A. , Braverman Richard , Mcduffie Elwood , Cooper Herbert L.
Published: March 2005

Expression of the tropomyosin‐1 isoform was enhanced by cDNA transfer in non‐transformed murine 3T3 fibroblasts and also in v‐Ki‐ras transformed fibroblasts in which native tropomyosin‐1 expression had been reduced and tropomyosin‐2 synthesis virtually eliminated by action of the oncogene. The level of synthesis of insert‐derived tropomyosin‐1 was similar in normal and transformed transductants (3–5 times normal levels). The high level of insert‐derived tropomyosin‐1 expression resulted in a considerable increase in tropomyosin‐1 utilization in the cytoskeleton of transformed cells, but this expression still did not reach normal levels, suggesting an oncogene‐related inhibition of tropomyosin utilization. A large proportion of newly synthesized native tropomyosin‐1 in normal, unmodified fibroblasts appeared in homodimers which, upon prolonged incubation, were largely converted to the heterodimers. Excess tropomyosin‐1 derived from the inserted cDNA also appeared largely as the homodimer in both normal and transformed cells. This homodimer was utilized effectively in the formation of cytoskeletal structures but was partially converted to heterodimer by chain exchange. Under steady‐state conditions, approximately 33% of the cytoskeletal tropomyosin‐1‐containing dimers were homodimers, compared to approximately 10% in normal fibroblasts. The results show that the increased amount of tropomyosin‐1 homodimer entering the cytoskeleton under conditions of tropomyosin‐1 excess, results in an atypical microfilament composition. The effect of this excess of tropomyosin‐1 homodimers on stability or function of microfilament fibers remains to be determined. The results also confirm that the mechanisms of rapid homodimer formation with conversion to heterodimers by chain exchange, known from in vitro studies, also occur in vivo.
Purification and Partial Amino Acid Sequence of Fuctinin, an Endogenous Inhibitor of Fucosyltransferase Activities
Authors: Ruggiero‐Lopez Daniel , Manioc Claudine , Geourjon Christophe , Louisot Pierre , Martin Ambroise
Published: March 2005

A powerful endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, was purified to homogeneity from rat small‐intestinal mucosa. The purification scheme involved DEAE–cellulose ion‐exchange chromatography, ammonium sulfate fractionation, hexyl–agarose hydrophobic chromatography and size‐exclusion HPLC. Active native fuctinin has an isoelectric point of 4.55 and apparent molecular mass approximately 66 kDa, whereas a single protein band with a molecular mass of approximately 24 kDa was obtained by denaturing polyacrylamide gel electrophoresis, suggesting that fuctinin is an oligomeric protein. Two‐dimensional polyacrylamide gel electrophoresis displayed eight spots in this single band. Comparisons of the N‐terminal amino acid sequences of each spot support the idea of the existence of three related polypeptides and suggest a proteolytic N‐terminal cleavage despite the use of an efficient protease inhibitor throughout the purification. In spite of the presence of an N‐glycosylation site, fuctinin is not glycosylated. One of the three polypeptides, peptide 3, possesses two consensus sequences for phosphorylation and a consensus sequence for myristoylation. The sequences of fuctinin‐related peptides, especially peptide 3, exhibit high similarity to the N‐terminal domain of the Set protein and a putative human leukocyte antigen‐associated protein. The possible implications of these results are discussed.
The Synthesis of Fatty Acid Ethyl Ester by Carboxylester Lipase
Authors: Tsujita Takahiro , Okuda Hiromichi
Published: March 2005

Carboxylester lipase obtained from pig pancreas is associated with fatty acid ethyl ester synthase as judged by their elution in the same fraction from a heparin–Sepharose column, coprecipitations by antibody against purified carboxylester lipase and identical profiles of inhibition by diisopropyl fluorophosphate. Only one polypeptide of molecular mass 74–kDa in purified carboxylester lipase was labeled by immunostaining and affinity labeling with [3H]diisopropyl fluorosphate. Bovine serum albumin decreased the fatty‐acid‐ethyl‐ester‐synthesizing activity in a concentration‐dependent manner.

On incubation of purified carboxylester lipase with trioleylglycerol in an ethanol/water mixture, fatty acid ethyl ester was formed in the presence of a high concentration of bovine serum albumin. The acyltransfer activities from trioleylglycerol to ethanol (ethanolysis) were approximately 25–30 times higher than the acyltransfer activities to water (hydrolysis). When cholesterol was used as an acceptor, acyltransfer activity from trioleylglycerol to cholesterol (cholesterolysis) was also observed. We propose the following mechanism of faty acid ethyl ester formation from triacyl glycerol. The enzyme attacks triacyl glycerol forming an acyl‐enzyme intermediate, and during the deacylation process, alcohol binds to fatty acid as an acceptor. These results suggest that during lipid (triacyl glycerol) degradation, carboxylester lipase contributes to non‐oxidative ethanol metabolism in the intestinal lumen.
The Effect of the Asn82→Asp Mutation in Yeast Cytochrome c Peroxidase Studied by Proton NMR Spectroscopy
Authors: Satterlee James D. , Alam Steve L. , Mauro J. Matthew , Erman James E. , Poulos Thomas L.
Published: March 2005

Proton NMR studies of the mutant of baker's yeast cytochrome c peroxidase‐cyanide with the Asn 82→Asp mutation ([N82D]cytochrome c peroxidase‐CN) are presented and compared to the wild‐type enzyme. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme‐bound ligands. His52 is an important participant in the initial hydrogen peroxide decomposition step of cytochrome c peroxidase. In wild‐type cytochrome c peroxidase‐CN, His52 hydrogen bonds to the neighboring Am82 peptide carbonyl group and to heme‐coordinated cyanide. His52 thus manifests itself as an extensively hydrogen bonded histidinium moiety. The principal result from this study is the observation that three hyperfine‐shifted resonances disappear from the spectrum of [N82D] cytochrome c peroxidase‐CN compared to the wild‐type enzyme. All three absent resonances in [N82D]cytochrome c peroxidase‐CN belong to His52 and this leads to the conclusion that the result of the mutation has been elimination of the His52–Am82 and His52–heme‐coordinated cyanide hydrogen bonds.
Synthesis and Characterization of Desulfovibrio gigas Rubredoxin and Rubredoxin Fragments
Authors: Christensen Hans E. M. , Hammerstad‐Pedersen Jan M. , Holm Arne , Iversen Gitte , Jensen Marianne H. , Ulstrup Jens
Published: March 2005

The 52‐residue Desulfovibrio gigas rubredoxin peptide chain has been synthesized and a procedure for chain folding around iron(II) developed. The folded, stable synthetic rubredoxin can be subjected to purification, and reversibly oxidized and reduced. Ultraviolet/visible absorption and CD spectra of both forms show all the same features as native D. gigas rubredoxin, and the symmetric and asymmetric Fe‐S stretching bands in the resonance Raman spectrum can be identified. In addition, the matrix‐assisted laser desorption mass spectrum of a peptide sample exposed to trace amounts of iron is dominated by a peak at 5735 Da very close to the value for the calculated molecular mass. Details in the ultraviolet/visible bandshape and mass spectrum, however, indicate remaining impurities. In comparison, a previously synthesized 25‐residue rubredoxin fragment with the non‐conserved positions 13–35 and 51–52 omitted and Val5–Glu50 anchored via glycine folds gives the correct molecular mass and ultraviolet/visible spectrum, but is much more labile than the 52‐residue protein. This shows that non‐conserved residues are crucial in protein folding and that chemical metalloprotein synthesis offers alternative prospects to microbiological protein engineering.
A Misfolded but Active Dimer of Bovine Seminal Ribonuclease
Authors: Kim Jin‐Soo , Raines Ronald T.
Published: March 2005

Bovine seminal ribonuclease (BS‐RNase) is an unusual homolog of RNase A. Isolated from bulls as a dimer, BS‐RNase has special biological properties including anitspermatogenic, antitumor and immunosuppressive activities. The structural bases for these properties are unknown. Four forms of BS‐RNase were isolated after folding and air oxidation of the denatured and reduced protein produced in Escherichia coli: two dimers (M = M and M×I, where × signifies an active site composed of residues from both subunits) and two monomers (M and I). Considerable ribonuclease activity was generated by air oxidation of an equimolar mixture of two inactive mutant proteins ([H12D]BS‐RNase and [H119D]BS‐RNase) prepared by site‐directed mutagenesis. This activity came from a dimer (M×I) with a composite active site. 1H‐NMR spectroscopy revealed that this dimer contained one correctly folded subunit (M), and one incorrectly folded subunit (I). Form I, which is a poor catalyst, was activated by ribonuclease S‐protein, suggesting that the C‐terminal portion of I was not folded properly. Electrospray‐ionization mass spectrometry and sulfhydryl group titration indicated that I contains a single oxidized sulfhydryl group, which cannot participate in a disulfide bond. These results show that quaternary structure in BS‐RNase is attained by the initial formation of two monomers, M and I, which then combine with another M to form M = M and M×I, respectively. Adventitious oxidation can thus lead to the formation of a misfolded but active enzyme (M×I).