bluuebunny/crossref_metadata_2025 huggingface dataset:
Autophosphorylation of type 2 casein kinase TS at both its α‐ and β‐subunits
Authors: Meggio Flavio , Brunati Anna Maria , Pinna Lorenzo A.
Published: July 2002

Rat liver casein kinase TS (Ck‐TS) having quarternary structure α2β2, autophosphorylates at its 25 kDa, β‐subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck‐TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β‐subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β‐subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β‐subunit, while promoting the autophosphorylation of the 38 kDa α‐subunit. In the presence of such polyarginyl effectors the 130 kDa Ck‐TS is converted into forms with higher sedimentation coefficient.
Tumour‐host metabolic interaction and cachexia
Authors: Lazo Pedro A.
Published: July 2002

Cachexia is a terminal metabolic problem observed in a wide variety of tumours. In this article I propose that the syndrome is a direct consequence of the common feature to all malignant tumours: growth. I suggest that the requirement for essential amino acids can be used as the unifying principle that links the tumour to the two main components of cachexia: muscle wastage and anorexia. This underlying factor is usually clouded by the overlapping of individual tumour characteristics.
t‐Butyl‐4‐hydroxyanisole, a novel respiratory chain inhibitor
Authors: Aldunate J. , Ferreira J. , Letelier M.E. , Repetto Y. , Morello A.
Published: July 2002

t‐Butyl‐4‐hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level ofcytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.
Cyclic GMP phosphodiesterase from cattle retina
Authors: Ovchinnikov Yu.A. , Lipkin V.M. , Kumarev V.P. , Gubanov V.V. , Khramtsov N.V. , Akhmedov N.B. , Gubanov V.E. , Muradov K.G.
Published: July 2002

The primary structure of the γ‐subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme γ‐subunit contains 87 amino acid residues, its N‐terminal amino group being acetylated.
Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S
Authors: Isemura Satoko , Saitoh Eiichi , Sanada Kazuo
Published: July 2002

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM‐Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.
Characterization of a cDNA coding for sex steroid‐binding protein of human plasma
Authors: Que Benito G. , Petra Philip H.
Published: July 2002

A cDNA (912 nucleotides) coding for human plasma sex steroid‐binding protein (SBP) was characterized from a phage clone previously isolated by screening a Charon 21A human liver cDNA library with rat androgen binding protein (ABP) cDNA. The deduced amino acid sequence from the cDNA indicated that the insert was a partial clone coding for 281 amino acids starting with residue 92 (glycine) encompassing the alternating leucyl residues and the carboxyl‐end 373 (histidine) as previously reported [(1986) Biochemistry 25, 7584]. The potential polyadenylation signal sequence ATTAAA is present as part of the 3′‐coding region and the stop codon TAA. Both are followed by a short 20 untranslated nucleotides and a poly(A) tract of 49 nucleotides. Significant homologous sequences (76%) at the DNA level exist between human SBP and rat ABP which might suggest the possibility that both evolved from a common primordial gene. Demonstration of the presence of an SBP cDNA in a human liver cDNA library provides the first evidence that liver is the site of SBP biosynthesis.
A ferriprotoporphyrin IX‐chloroquine complex promotes membrane phospholipid peroxidation A possible mechanism for antimalarial action
Authors: Sugioka Yumiko , Suzuki Mamoru , Sugioka Katsuaki , Nakano Minoru
Published: July 2002

The effect of a ferriprotoporphyrin IX ‐chloroquine complex on phospholipid membranes was investigated on the basis of iron‐induced lipid peroxidation using microsomal phospholipid liposomes. The ferriprotoporphyrin IX ‐chloroquine complex remarkably promoted peroxidative cleavage of unsaturated phospholipids in liposomes, compared with ferriprotoporphyrin IX alone. Neither the combination of protoporphyrin IX with chloroquine nor the combination of Fe 3+‐ADP with chloroquine promoted lipid peroxidation. DL‐α‐Tocopherol completely inhibited lipid peroxidation induced by the ferriprotoporphyrin IX ‐chloroquine complex.
Amino acid sequence of iron‐superoxide dismutase from Pseudomonas ovalis
Authors: Isobe Toshiaki , Fang Yan-Il , Muno Daisaku , Okuyama Tsuneo , Ohmori Daijiro , Yamakura Fumiyuki
Published: July 2002

The amino acid sequence of iron‐superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and diluted acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated M
r of 21 421. the sequence is highly homologous (65% identity) to the recently published sequence of the iron‐superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese‐superoxide dismutase by sharing 33–53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X‐ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His‐26, His‐74, Asp‐156 and His‐160, which align with the ligands to the manganese in the Thermus thermophilus manganese‐superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X‐ray crystallographic data that are now available at 2.9 Å resolution.
A mechanism for accelerated degradation of intracellular proteins after limited damage by free radicals
Authors: Dean Roger T.
Published: July 2002

I propose that limited free radical attack upon proteins, occurring continuously in cells, creates new N‐termini (notably aspartate and glutamate) which render the proteins more susceptible to proteolysis by the ubiquitin conjugation system. I suggest that these reactions are a significant part of the previously described ‘N‐end’ and ‘PEST’ rules, which indicate amino acid termini or sequences which tend to dictate short protein half‐lives. I also argue that the N‐end rule may apply to sequestered intracellular sites, such as mitochondria, these also being sites of radical generation.
A TY1 element is inserted in the CYR1 control region of Saccharomyces cerevisiae strain AB320
Authors: Lenzen G. , Masson P. , Jacquemin J.-M. , Danchin A.
Published: July 2002

Southern blotting using a 5′‐proximal probe of the Saccharomyces cerevisiae CYR1 gene has revealed heterogeneity in laboratory strains. It is demonstrated that strain AB320 contains a Ty1 element inserted in the promoter region of CYR1. The Ty1 orientation suggests that transcription of CYR1 is initiated downstream from the insertion region.
Hydrophobic 3.7 kDa surfactant polypeptide: Structural characterization of the human and bovine forms
Authors: Johansson Jan , Jörnvall Hans , Eklund Anders , Christensen Niels , Robertson Bengt , Curstedt Tore
Published: July 2002

The human and bovine forms of the hydrophobic 3.7 kDa surfactant polypeptide have been structurally analyzed. The polypeptide is essentially inert to enzymatic proteolysis, and methods for analysis include peptide handling in organic solvents and fragment generation by limited acid hydrolysis. The molecule exhibits N‐terminal trimming, and the relative abundance of the different starting positions varies both among species and between adult and fetal forms of the surfactant polypeptide. The bovine major form is one residue shorter than the mature 35‐residue human molecule. Comparison of the porcine, human and bovine polypeptides reveals a conserved hydrophobic middle/C‐terminal segment and a variable hydrophilic N‐terminal part.
Immunoprecipitation of human adrenal microsomal antigen
Authors: Furmaniak J. , Talbot D. , Reinwein D. , Benker G. , Creagh F.M. , Smith B.Rees
Published: July 2002

Human adrenal microsomes have been labelled with 125I and immunoprecipitated with sera from patients with Addison's disease. The immunoprecipitates were then analysed by SDS‐PAGE and autoradiography. 13 of the 23 sera from the Addison patients studied contained antibodies which reacted with a 55 kDa adrenal microsomal protein. The same 13 sera were also positive for adrenal antibodies as judged by immunofluorescence. The 55 kDa protein was not immunoprecipitated from placenta or thyroid microsomes by Addison sera. Furthermore, patients with Graves' disease or rheumatoid arthritis did not immunoprecipitate the 55 kDa protein from adrenal microsomes. Our studies suggest therefore that Addison sera contain antibodies to a 55 kDa adrenal specific protein which may well be the antigen observed on immunofluorescence.
Resonance Raman evidence for an unusually strong exogenous ligand—metal bond in a monomeric nitrosyl manganese hemoglobin
Authors: Lin Shun-Hua , Yu Nai-Teng , Gersonde Klaus
Published: July 2002

Resonance Raman spectroscopy has been employed to determine the vibrational modes of monomeric nitrosyl manganese Chironomus thummi thummi hemoglobin (CTT IV). This insect hemoglobin has no distal histidine. By applying various isotope‐labeled nitric oxides (14N16O, 15N16O, 14N18O), we have identified the Mn II‐NO stretching mode at 628 cm−1, the MnII‐N‐O bending mode at 574 cm−1 and the N‐O stretching mode at 1735 cm−1. The results suggest a strong MnII◀NO bond and a weak N◀O bond. The vinyl group substitution does not influence the ν(MnII‐NO), δ(MnII‐N‐O) and ν(N◀O) vibrations. The MnII‐NO stretching frequency is insensitive to distal histidine interactions with NO, whereas the N◀O stretching frequency is sensitive. Nitric oxide also binds to Met manganese CTT IV to form an MnIII·NO complex which undergoes a slow but complete autoreduction resulting in the MnII·NO species. In manganese meso‐IX CTT IV, the MnIII·NO MnII·NO conversion alters the intensities of the porphyrin ring modes at 342, 360, 1587 and 1598 cm−1, but shifts the frequencies at 1504 and 1633 cm−1 (in MnIII·NO) to 1497 and 1630 cm−1 (in MnII·NO), respectively. The unshifted marker line at 1378 cm−1 reflects the fact that the π* electron densities of the porphyrin ring are the same in the two complexes.
Synthesis of Saccharomyces cerevisiae invertase by Schizosaccharomyces pombe
Authors: Sánchez Yolanda , Moreno Sergio , Rodríguez Luis
Published: July 2002

In order to gain information on the ability of the glycosylation system of Schizosaccharomyces pombe to process heterologous glycoproteins, the expression of Saccharomyces cerevisiae invertase in the former yeast was studied. Sc. pombe cells are able to produce enzymatically active invertase from the S. cerevisiae SUC2 gene introduced by transformation and the enzyme is glycosylated and secreted into the cell wall. However, Sc. pombe transformants do not glycosylate the heterologous enzyme as their own invertase since it is not bound by the lectin from Bandeiraea simplicifolia seeds, which indicates the absence of terminal galactose residues. Moreover, the electrophoretic mobility of the heterologous invertase is similar to that of the large enzyme from S. cerevisiae, both in its native form and after being deglycosylated with Endo H. These results suggest that the polypeptide chain of S. cerevisiae invertase is the primary factor for the glycosylation in Sc. pombe cells.
α‐Melanocyte stimulating hormone promotes neurite outgrowth in chromaffin cells
Authors: Demeneix Barbara , Grant Nancy J.
Published: July 2002

Chromaffin cells from adult bovine adrenal medulla were found to develop neurites when cocultured with pituitary intermediate lobe (IL) cells. In coculture 51.7% of the chromaffin cells extended neurites compared with 12% in control cultures (chromaffin cells alone). A soluble factor released by IL cells was apparently involved as medium conditioned by contact with IL cells also promoted neurite outgrowth. Moreover, the addition of αMSH, one of the pro‐opiomelanocortin‐derived peptides secreted by IL cells, alone reproduced this effect in a dose‐dependent manner. The data provide evidence for a neurotrophic role of αMSH.
Limited proteolysis of tumor cells increases their plasmin‐binding ability
Authors: Camacho Mercedes , Fondaneche Marie-Claude , Burtin Pierre
Published: July 2002

Mild proteolytic treatment of SW 1116 tumor cells with trypsin or plasmin increases their plasmin‐binding ability considerably by increasing the number of binding sites without altering their affinity. This mechanism may be operative for increasing the concentration of active plasmin at the surface of tumor cells. C‐terminal lysine residues are involved in plasmin binding to cells, since treatment of cells with carboxypeptidase B decreases this binding by 50%.
Modification of a catalytically important residue of indoleglycerol‐phosphate synthase from Escherichia coli
Authors: Eberhard Marc , Kirschner Kasper
Published: July 2002

The active‐site residues of indoleglycerol‐phosphate synthase from Escherichia coli were tentatively localized by comparing crystallographic data with the amino acid identities among the known indoleglycerol‐phosphate synthase sequences. To test the validity of the resulting model of catalysis one of the residues in the presumptive active site, Lys 55, was changed to serine using oligonucleotide‐directed mutagenesis. The specificity constant k
cat/K
m of the mutant is 3 × 104‐times lower than that of the wild‐type enzyme, due to a 60‐fold decrease in k
cat and a 450‐fold increase in K
m. This finding shows that Lys 55 is important for both catalysis and substrate binding.
Dual effects of G‐protein activation on Ca‐dependent exocytosis in bovine lactotrophs
Authors: Sikdar S.K. , Zorec R. , Brown D. , Mason W.T.
Published: July 2002

The whole‐cell patch‐clamp technique was used to measure cell membrane capacitance (C
m) to monitor exocytosis in single‐cultured bovine prolactin‐secreting cells (lactotrophs) of the anterior pituitary. The cells were dialyzed with solutions containing different concentrations of ionised Ca and non‐hydrolyzable GTP analogues (GTP‐γ‐S and GMP‐PNP) to activate G‐proteins. We have identified two distinct effects of G‐protein activation on Ca‐induced exocytosis: (i) the maximum C
m increase due to intracellular Ca‐dependent exocytosis was diminished, suggesting an inhibitory role of G‐proteins close to the site of granule fusion, while (ii) the rate of C
m increase (ΔC
m/Δt) was facilitated, revealing conversely a stimulatory role of G‐proteins in the translocation of secretory granules to the fusion sites.
Neuropeptide Y‐induced intracellular Ca2+ increases in vascular smooth muscle cells
Authors: Mihara Shin-ichi , Shigeri Yasushi , Fujimoto Masafumi
Published: July 2002

The effect of neuropeptide Y (NPY) on cytosolic free Ca2+ concentration ([Ca2+],) was studied in cultured smooth muscle cells from porcine aorta (PASMC) and compared with the effect of bradykinin (BK) and angiotensin II (ATII) on [Ca2+. All peptides induced dose‐dependent and transient rises in [Ca2+], which were not blocked by extracellular EGTA, but the NPY response was different from the others' as follows. First, the [Ca2+]; rise induced by NPY was not as rapid as that induced by BK or ATII. Second, pertussis toxin abolished the [Ca2+], rise induced by NPY, but not by BK or ATII. Third, following initial treatment with BK, PASMC were able to respond to NPY, but not to ATII. Finally, BK and ATII, but not NPY, significantly increased inositol 1,4,5‐trisphosphate (Ins(l,4,5)P3) generation. Although NPY attenuated forskolin‐induced accumulation of cyclic AMP, forskolin‐ and 3‐isobutyl‐1‐methyl‐xanthine‐induced alterations in intracellular cyclic AMP did not affect the NPY‐induced [Ca2+]i rise. These results suggest that'NPY increases [Ca2+], by a pertussis toxin‐sensitive GTP binding protein‐involved mechanism which is not mediated by the intracellular messengers such as Ins(1,4,5)P3 and cyclic AMP.
Two different forms of endothelin receptors in rat lung
Authors: Masuda Yasushi , Miyazaki Hitoshi , Kondoh Motohiro , Watanabe Hirotoshi , Yanagisawa Masashi , Masaki Tomoh , Murakami Kazuo
Published: July 2002

We provided evidence for the presence of two distinct types of rat endothelin (ET) receptors having different molecular masses by affinity labelling of rat lung membranes with 125I‐ET‐1, ET‐2 and ET‐3 and SDS‐PAGE followed by autoradiography. One type, which is 44 kDa has a higher affinity for ET‐1 and ‐2 than ET‐3, whereas the other type, which is 32 kDa, preferably interacts with ET‐3 rather than ET‐1 and ‐2.
Modulation ofinterleukin‐1β production by cyclic AMP in human monocytes
Authors: Hurme Mikko
Published: July 2002

Elevation of cAMP has been considered to be an important downregulative signal in the production ofinterieukin‐1(IL‐1). This study demonstrates that this phenomenon is dependent on the signal used to activate the IL‐1 production. The IL‐1β production of lipopolysaccharide activated human monocytes was readily inhibited by dibutyryl cAMP. This took place without a significant change in the steady‐state levels of IL‐1β mRNA. By contrast, in PMA activated monocytes 100 μM dibutyryl cAMP increased in IL‐1β production ca. 4‐fold. The steady‐statelevels of IL‐1β mRNA were also simultaneously increased.
Time‐resolved fluorescence studies on mutants of the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: Schulze Egbert , Westphal Adrie H. , Berg Axel , de Kok Arie
Published: July 2002

Fluorescence anisotropy decays were measured for the wild‐type dihydrolipoyl transacetylase (E2) component of pyruvate dehydrogenase complex from Azotobacter vinelandii and E. coli and for E2‐mutants from A. vinelandii in which the alanine‐proline‐rich sequence between the binding domain and the catalytic domain is partially or completely deleted. In both E2‐mutants the rotational mobility of the lipoyl domain and the overall activity after reconstitution of the complex are significantly decreased indicating the important role of the deleted sequence for the movement of the lipoyl domain and the transfer of substrates between the different active sites within the complex.
Immunological detection of the mitochondrial F1‐ATPase α subunit in the matrix of rat liver peroxisomes
Authors: Cuezva J.M. , Santarén J.F. , González P. , Valcarce C. , Luis A.M. , Izquierdo J.M.
Published: July 2002

Rat liver peroxisomes contain in their matrix the α‐subunit of the mitochondrial F1‐ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No β‐subunit of the mitochondrial F1‐ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala‐Lys‐Leu) is found at the carboxy terminus of the mature α‐subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the α‐subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713‐7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.
Comparison of structures of dry and wet hen egg‐white lysozyme molecule at 1.8 Å resolution
Authors: Kachalova G.S. , Morozov V.N. , Morozova T.Ya. , Myachin E.T. , Vagin A.A. , Strokopytov B.V. , Nekrasov Yu.V.
Published: July 2002

A high resolution structure of hen egg‐white lysozyme containing 36 ± I mol H2O per mol of protein has been obtained using triclinic (P1) crystals cross‐linked with glutaraldehyde. Analysis of dehydration‐induced structural changes has revealed displacement in relative position of domains and numerous small displacements in positions of individual atoms with r.m.s. deviation of main atoms 0.60 Å, and that of all atoms 0.97 Å. An increase in the average packing density of atoms in dry lysozyme by 4–6% seems to be the most probable reason for the loss of its activity and mobility.
The presence of two types of prorenin converting enzymes in the mouse submandibular gland
Authors: Kim Won-Sin , Nakayama Kazuhisa , Murakami Kazuo
Published: July 2002

We have recently demonstrated, by protein and cDNA sequence analyses, that prorenin converting enzyme (PRECE) in the ICR mouse submandibular gland is identical to the epidermal growth factor‐binding protein (EGF‐BP) type B. the mGK‐13 gene product identified in Balb/c mouse. However. in the course of cDNA cloning, we noticed the presence of the other cDNA type highly homologous but not identical to the PRECE cDNA. The sequence of the newly identified cDNA was identical to that of the pSGP‐2 cDNA cloned from NMRI mice, which also encodes EGF‐BP type B different at 9 out of 261 amino acids from the mGK‐13 product. Although this difference has been explained by strain polymorphism, our results indicate that these two proteins are distinct gene products. The product of the newly identified cDNA also had a prorenin converting activity. Thus, the products of both cDNAs identified in previous and present studies are involved in maturation of two bioactive polypeptides, renin and EGF.
The modulation of ferryl myoglobin formation and its oxidative effects on low density lipoproteins by nitric oxide
Authors: Dee Gareth , Rice-Evans Catherine , Obeyesekera Sharmin , Meraji Shokoufeh , Jacobs Michael , Bruckdorfer K.Richard
Published: July 2002

This study has investigated the interactions between nitric oxide and haem protein radicals. The results demonstrate that nitric oxide interacts with activated ferrylmyoglobin species with reduction to metmyoglobin, but the extent and duration of the reduction depends on the relative concentrations of nitric oxide and hydrogen peroxide. Ferryl myoglobin has a much greater relative potential for oxidising polyunsaturated fatty acid side chains in low density lipoproteins than in cell membranes. The peroxidative response can be modulated by nitric oxide: ferryl myoglobin‐mediated peroxidation of LDL may be enhanced or suppressed by nitric oxide depending on the relative concentrations of NO and hydrogen peroxide.
Effects of substitution of aspartate‐440 and tryptophan‐487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis
Authors: Diefenbach Russell J. , Candy Judith M. , Mattick John S. , Duggleby Ronald G.
Published: July 2002

A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site‐directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half‐saturating concentrations increasing from 0.64 to 9.0 μM for thiamin diphosphate and from 4.21 to 45 μM for Mg2+. Unlike the wild‐type enzyme, there was little quenching of tryptophan fluorescence upon adding, cofactors to this modified form. The data suggest that tryptophan‐487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine of glycine for aspartate‐440, a residue which is conserved between many thiamin diphosphate‐dependent enzymes, completely abolishes enzyme activity.
Yeast ADP/ATP carrier (AAC) proteins exhibit similar enzymatic properties but their deletion produces different phenotypes
Authors: Drgon̆ T. , S̆abová L. , Gavurniková G. , Kolarov J.
Published: July 2002

Saccharomyces cerevisiae strains expressing a single type of ADP/ATP carrier (AAC) protein were prepared from a mutant in which all AAC genes were disrupted, by transformation with plasmids containing a chosen AAC gene. As demonstrated by measurements of [14C]ADP specific binding and transport, all three translocator proteins, AAC1, AAC2 and AAC3 when present in the mitochondrial membrane, exhibited similar translocation properties. The disruption of some AAC genes, however, resulted in phenotypes indicating that the function of these proteins in whole cells can be quite different. Specifically, we found that the disruption of AAC1 gene, but not AAC2 and AAC3, resulted in a change in colony phenotype.
Isolation from Spirulina membranes of two photosystem I‐type complexes, one of which contains chlorophyll responsible for the 77 K fluorescence band at 760 nm
Authors: Shubin V.V. , Bezsmertnaya I.N. , Karapetyan N.V.
Published: July 2002

Two types of chlorophyll‐protein complexes of photosystem 1 (PSIa, PSIc) have been isolated from the membranes of Spirulina platensis using a Triton X‐100 treatment and chromatography on DEAE‐Toyopearl. The complexes are equally enriched with P700 (Chl: P700=100–110) but show different electrophoretic molecular masses ‐ 140 (PSIa) and 320 kDa (PSIc) ‐ and differ in the content of long‐wavelength absorbing Chl. PSIa has a typical PSI fluorescence band at 730 nm (F730) as the main band at 77 K, whereas PSIc is responsible for F760, the intensity of which depends on the redox state of P700. PSIc only shows 77 K light‐induced variable fluorescence at 760 typical of Spirulina membranes and cells.
A carboxyl‐terminal truncated version of the activin receptor mediates activin signals in early Xenopus embryos
Authors: Nishimatsu S. , Iwao M. , Nagai T. , Oda S. , Suzuki A. , Asashima M. , Murakami K. , Ueno N.
Published: July 2002

The function of a carboxyl‐terminal truncated version of the Xenopus activin receptor, encoded by a previously isolated gene XSTK2, was investigated in early embryos. The transcript corresponding to the truncated receptor gene was detected throughout embryonic development although the temporal expression pattern was different from that of an intact receptor. Injection of X5TR2 mRNA into early embryos resulted in the formation of a duplicated body axis. Mesoderm induction as evaluated by the activation of the α‐actin gene in presumptive ectoderm (animal cap) treated with exogenous activin was significantly enhanced by the injection of XSTK2 mRNA. These results suggest that the truncated receptor is capable of transmitting the activin signal to the same extent as the native receptor.
Trimethylamine dehydrogenase of bacterium W3A1 Molecular cloning, sequence determination and over‐expression of the gene
Authors: Boyd Geoffrey , Mathews F.Scott , Packman Leonard C. , Scrutton Nigel S.
Published: July 2002

The gene encoding trimethylamine dehydrogenase (EC 1.5.99.7) from bacterium W3A1 has been cloned. Using the polymerase chain reaction a 530 bp DNA fragment encoding a distal part of the gene was amplified. Using this fragment of DNA as a probe, a clone was then isolated as a 4.5 kb BamHI fragment and shown to encode residues 34 to 729 of trimethylamine dehydrogenase. The polymerase chain reaction was used also to isolate the DNA encoding the missing N‐terminal part of the gene. The complete open reading frame contained 2,190 base pairs coding for the processed protein of 729 amino acids which lacks the N‐terminal methionine residue. The high‐level expression of the gene in Escherichia coli was achieved by the construction of an expression vector derived from the plasmid pKK223‐3. The cloning and sequence analysis described here complete the partial assignment of the amino acid sequence derived from chemical sequencing (1) and will now permit the refinement of the crystallographic structure of trimethylamine dehydrogenase and also a detailed investigation of the mechanism and properties of the enzyme by protein engineering.
Expression of retinoic acid nuclear receptors in the mouse embryonal carcinoma cell line PCC7‐Mz1
Authors: Heiermann Reinhard , Rentrop Martin , Lang Elke , Maelicke Alfred
Published: July 2002

Mouse embryonal carcinoma cell line PCC7‐Mz1 can serve as a model of mammalian neural development [1989, J. Cell. Biol. 109, 2481–2493]. Upon exposure to all‐trans retinoic acid (RA), Mz1 cells differentiate into a stable pattern of neurons, astroglia and fibroblasts whereas variants of the parental cell line either are restricted in their patterns of derivatives or do not respond at all to RA. Using gene probes specific for the α1, α2 and β2 isoforms of the retinoic acid nuclear receptor, we have studied by Northern blot analysis the expression of these transcription factors in uninduced and induced cells of clone Mz1 and in variants with different developmental potential. α1‐RAR is expressed constitutively in all variants independent of whether RA is present or not. Soon after addition of 10−7 M RA, α2‐RAR is induced in RA‐responsive cells reaching within a few hours a plateau level that remains unchanged throughout the developmental process. In contrast, the β2 isoform is expressed only transiently after RA‐induction despite the continuous presence of RA. Other RAR isoforms are expressed only in trace amounts
Inhibition of passive cutaneous anaphylaxis by synthetic human immunoglobulin E peptide fragments
Authors: Nio Noriki , Seguro Katsuya , Ariyoshi Yasuo , Ishii Katsumi , Nakamara Hideo
Published: July 2002

In an attempt to determine the regions responsible for type 1 immediate hypersensitivity, a total of 42 peptide fragments, which cover the CH3–CH4 domains in human immunoglobulin E (IgE), were chemically synthesized. Several peptide fragments located in the amino acid sequences Ala329—Thr357 and Arg119—Ala463, inhibited passive cutaneous anaphylaxis (PCA) in vivo. In order to pinpoint the sites responsible for the inhibition of the PCA reaction, various rragment peptides in these two regions were synthesized. As a result, residues Pro343—Leu348, Pro426—Thr433, and Ser456—Thr401 were suggested to be involved in type I immediate hypersensitivity.
Diversion of the sign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs
Authors: Takahashi Tetsuo , Kubota Mameru , Watanabe Masakatsu , Yoshihara Kazuo , Derguini Fadila , Nakanishi Koji
Published: July 2002

The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtil is negatively phototactic in the presence of the native chromophore all‐trans retinal. In contrast, analog chromophores such as a ring‐acyclic retinal and those in which trans/cis isomerization about the C11–C12 double bond was blocked induced predominantly positive pho totaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted Hypothesis [1980, Microbiol. Rev. 44, 572–630] on phototaxis mechanisms.
Phosphorylation of a phosphoinositidase C‐linked muscarinic receptor by a novel kinase distinct from β‐adrenergic receptor kinase
Authors: Tobin A.B. , Keys B. , Nahorski S.R.
Published: July 2002

Muscarinic receptor kinase activity previously described in intact CHO cells transfected with human m3‐muscarinic receptor cDNA (CHO‐m3 cells) [Tobin, A.B and Nahorski, S.R (1993) J. Biol. Chem. 268,9817\3‐9823] was found to be associated, at least in part, with a crude membrane fraction of CHO‐m3 cell lysates. Phosphorylation of the m3‐muscarinic receptor was agonist dependent, reaching a maximum after 10 min exposure to carbachol (1 mM) and was completely blocked by atropine (10μM). m3‐Muscarinic receptor phosphorylation was insensitive to Zn2+ (0.1 mM) and heparin (1 ), concentrations that inhibit endogenous β‐adrenergic receptor kinase activity present in CHO‐m3 cells strongly suggesting that the m3‐muscarinic receptor kinase is distinct fromβ‐adrenergic receptor kinase. A role for protein kinase C can also be eliminated on the basis that the potent protein kinase C inhibitor, Ro‐318220 (1μM), had no effect on agonist‐mediated m3‐muscarinic receptor phosphorylation. Further, the inability of calcium (300μM), cAMP (0.2 mM) and cGMP (0.2 mM) to elevate the basal phosphorylation state of m3‐muscarinic receptors eliminates a role for protein kinases regulated by these second messengers. Finally, agonist mediated phosphorylation appears to be independent of G‐protein activation as both GDP‐β‐S (500μM) and GTP‐γ‐S (100μM) did not influence m3‐muscarinic receptor phosphorylation.
γ‐Glutamylcysteinylglutamic acid ‐ a new homologue of glutathione in maize seedlings exposed to cadmium
Authors: Meuwly Philippe , Thibault Pierre , Rauser Wilfried E.
Published: July 2002

Exposure of plants to Cd induces the appearance of several thiols based on glutathione and known as class III metallothioneins (or phytochelatins). A new tripeptide with the structure γ‐GluCysGlu accumulated in roots and shoots of Cd‐exposed maize seedlings. This thiol was purified and identified by tandem mass spectrometry. The fragmentation pattern of the maize tripeptide was identical to that of the synthetic compound. Like glutathione, this new tripeptide may serve as a precursor for longer‐chain peptides involved in metal detoxification through the formation of Cd‐binding complexes.
The NMR determination of the IIAmtl binding site on HPr of the Escherichia coli phosphoenol pyruvate‐dependent phosphotransferase system
Authors: van Nuland Nico A.J. , Kroon Gerard J.A. , Dijkstra Klaas , Wolters Gea K. , Scheek Ruud M. , Robillard George T.
Published: July 2002

The region of the surface of the histidine‐containing protein (HPr) which interacts with the A domain of the mannitol‐specific Enzyme II (IIAmtl) has been mapped by titrating the A‐domain into a solution of 15N‐labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty‐five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of IIAmtl while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of IIAmtl resulted in chemical shift changes in a sub‐set of the above residues; these were located more in the vicinity of the active site phospho‐histidine. Phosphorylation of the HPr/IIAmtl complex resulted in a HSQC spectrum which was indistinguishable from the P‐HPr spectrum in the absence of IIAmtl indicating that, as expected, the complex P‐HPr/P‐IIAmtl does not exist even at the high concentrations necessary for NMR.
Cell‐penetrating inhibitors of calpain block both membrane fusion and filamin cleavage in chick embryonic myoblasts
Authors: Kwak Kyu Bong , Kambayashi Jun-ichi , Kang Man Sik , Ha Doo Bong , Chung Chin Ha
Published: July 2002

Benzyloxycarbonyl(Z)‐Leu‐nLeu‐H (calpeptin) and Z‐Leu‐Met‐H, cell‐penetrating inhibitors of calpain, were found to block myoblast fusion without any effect on cell proliferation and alignment along their bipolar axis. They also inhibited the accumulation of creatine kinase during myogenesis. These effects were dose‐dependent, and could be reversed upon removal of the drug from the culture medium. Furthermore, treatment of the inhibitors prevented the hydrolysis of filamin, which is sensitive to cleavage by calpain in vitro and interferes with actin‐myosin filament formation by cross‐linking F‐actin molecules. On the other hand, leupeptin, which can also inhibit calpain in vitro but can not penetrate into cells, showed little or no effect on both myoblast fusion and filamin cleavage. These results suggest that calpain may play an important role in cytoskeletal reorganization that is requisite for myoblast fusion. The role of calpain on the expression of muscle‐specific proteins remains unknown.
The low‐temperature folding intermediate of hyperthermophilic d‐glyceraldehyde‐3‐phosphate dehydrogenase from Thermotoga maritima shows a native‐like cooperative unfolding transition
Authors: Rehaber Verena , Jaenicke Rainer
Published: July 2002

Hyperthermophilic d‐glyceraldehyde‐3‐phosphate dehydrogenase from Thermotoga maritima, after denaturation in 6 M guanidinium chloride and subsequent renaturation by dilution at 3°C, forms a ‘low‐temperature intermediate’ with its native quaternary structure and most of its dichroic absorption restored, but with significant differences in its fluorescence properties compared to those of the native enzyme. Shifting the temperature beyond 10°C, the enzyme is reconstituted to high yields and to an overall structure indistinguishable from the initial native state [FEBS Lett. 290 (1991) 235‐238]. These criteria suggest that the cold intermediate represents an ‘assembled molten globule’. However, present equilibrium transition data prove the cold intermediate to be native‐like, in that it exhibits a reversible highly cooperative conformational transition to the unfolded state which is incompatible with the typical characteristics of the molten globule state of globular proteins.
Membrane interaction of Escherichia coli penicillin binding protein 5 is modulated by the ectomembranous domain
Authors: Phoenix David A. , Pratt Julie M.
Published: July 2002

E. coli penicillin binding protein (PBP) 5 is anchored to the periplasmic face of the inner membrane by a C‐terminal domain which is predicted to form an amphipihilic α‐helix. Here we show that the presence of a substrate analogue, benzyl penicillin, causes the protein to be converted from a membrane bound urea inaccessible form to a urea extractable form. If the anchor region is fused to the periplasmic protein, β‐lactamase, the fusion protein becomes membrane bound but is unable to exhibit the changes in urea extractability which are observed with PBP5. We therefore conclude that although the C‐terminus of PBP5 is sufficient to anchor the protein to the membrane surface the ectomembranous domain can affect the state of the anchor and in vivo changes in the state of anchoring may be related to enzyme activity.
Mosses do express conventional, distantly B‐type‐related phytochromes phytochrome of Physcomitrella patens (Hedw.)
Authors: Kolukisaoglu H.Üner , Braun Birgit , Martin William F. , Schneider-Poetsch Hansjörg A.W.
Published: July 2002

We have screened a cDNA library of the moss Physcomitrella patens (Hedw.) for phytochrome sequences. The isolated sequences turned out to encode a phytochrome dissimilar to the phytochrome type postulated for the moss Ceratodon [(1 992) Plant Mol. Biol. 20, 1003–1017] Physcomitrella phytochrome was completely alignable to fern phytochrome (Selaginella) and phytochromes of higher plants. The frequency of clones encoding this phytochrome indicated that a Ceratodon‐like type should only be expressed, if at all, with lower frequencies than the sequenced phytochrome cDNA. Sequence differences between lower plant phytochromes are small as compared to phytochrome types of higher plants.
Purification of dystrophin‐related protein (utrophin) from lung and its identification in pulmonary artery endothelial cells
Authors: Matsumura Kiichiro , Shasby Douglas M. , Campbell Kevin P.
Published: July 2002

Dystrophin‐related protein (DRP or utrophin) is an autosomal homologue of dystrophin, a membrane cytoskeletal protein encoded by the Duchenne muscular dystrophy gene. In contrast to dystrophin which is predominantly expressed in muscle, DRP is expressed in various tissues. Here we report the purification and biochemical characterization of DRP from lung which shows the highest levels of DRP expression among adult tissues. DRP was purified from the high alkaline extract of lung membranes using heparin‐agarose column chromatography followed by anti‐DRP immunoaffinity column chromatography. DRP was expressed in the cultured pulmonary artery endothelial cells. Expression of DRP in endothelial cells could explain its abundant expression in lung. In analogy to dystrophin of muscle cells, DRP could be playing an important role in the mechanical stress mechanisms of endothelial cells.
Activation of human peripheral monocytes by angiotensin II
Authors: Hahn Alfred W.A. , Jonas Uwe , Bühler Fritz R. , Resink Thérèse J.
Published: July 2002

This study has investigated the ability of the vasoconstrictor peptide angiotensin II to activate human peripheral blood monocytes. Activation was monitored by measuring both the release of tumor necrosis factor α from monocytes and their adhesion to monolayers of human endothelial cells. Angiotensin II‐elicited activation of monocytes was dose‐dependent (half‐maxiinally effective concentration ≈ 0.2 nM), saturable (maximally effective concentration ≈ 5 nM), and sensitive to inhibition by the angiotensin type 1 receptor antagonist ZD 7155. Such direct actions imply that angiotensin II is an important candidate stimulus for the subendothelial infiltration of monocytes observed in atherogenesis and hypertension.
Comparison of kinetic properties between MSS4 and Rab3A GRF GDP/GTP exchange proteins
Authors: Miyazaki Akira , Sasaki Takuya , Araki Keishi , Ueno Nozomi , Imazumi Katsunori , Nagano Fumiko , Takahashi Kazuo , Takai Yoshimi
Published: July 2002

The kinetic properties of MSS4 are studied in comparison with those of Rab3A GRF. MSS4 stimulates the dissociation of [3H]GDP from the lipid‐modified and lipid‐unmodified forms of Rab3A to the same extent, although Rab3A GRF is more effective on the lipid‐modified form than on the lipid‐unmodified form. Both MSS4 and Rab3A GRF are inactive on other Rab/Sec/Ypt family members including at least Rab2, Rab5, and Rab11. Rab GDI inhibits the MSS4 and Rab3A GRF effects on the lipid‐modified form of Rab3A, but the doses of Rab GDI necessary for this inhibitory effect on Rab3A GRF are lower than those on MSS4. Moreover, Rab GDI slightly inhibits the Rab3A GRF effect on the lipid‐unmodified form of Rab3A, but does not affect the MSS4 effect on the lipid‐unmodified form of Rab3A. These results suggest that MSS4 and Rab3A GRF are different GDP/GTP exchange proteins for Rab3A.
Hybrid proteins between Pseudomonas exotoxin A and poliovirus protease 2Apro
Authors: Novoa Isabel , Feduchi Elena , Carrasco Luis
Published: July 2002

Two hybrid proteins between Pseudomonas aeruginosa exotoxin A (PE) and poliovirus protease 2Apro have been generated. One hybrid protein contains the poliovirus 2Apro sequence replacing the region of PE corresponding to amino acids 413–607. The other hybrid contains in addition the transforming growth factor sequence. The two hybrid proteins were efficiently synthesized in E. coli cells using the inducible pET vectors. Both hybrid toxins cleaved p220 (eIF‐4γ) when the recombinant plasmids were transfected in COS cells infected with recombinant vaccinia virus bearing the T7 RNA polymerase gene.
Enzyme‐catalyzed uridine phosphorolysis: SN2 mechanism with phosphate activation by desolvation
Authors: Komissarov A.A. , Moltchan O.K. , Romanova D.V. , Debabov V.G.
Published: July 2002

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein‐5′‐isothiocyanate (fluorescein residue occupies an uridine‐binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54–58]) was selectively modified with irreversible inhibitor SA‐423, which reacts near the phosphate‐binding subsite. The double‐modified uridine phosphorylase is assumed to imitate the enzyme—substrate complex. Modification with SA‐423 was accompanied with dramatic changes in the absorption spectrum of active site‐linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.
Solvent and thermal denaturation of the acidic compact state of apomyoglobin
Authors: Sirangelo Ivana , Bismuto Ettore , Irace Gaetano
Published: July 2002

The stability of the acidic compact state of apomyoglobin toward the denaturant action of guanidinium hydrochloride and temperature was studied by examining the effects induced on the intrinsic tryptophanyl fluorescence and that of the adduct formed with 1,8‐anilinonaphthalenesulfonate (ANS). The results indicated that the disorganization of tryptophanyl environments is caused by a cooperative discrete molecular transition, thus contrasting the assumption that the acidic compact form of apomyoglobin might be a molten globule state. The unfolding of the ANS binding regions was found to involve, at least, two stages over a wide range of denaturant concentrations.
Anti‐idiotypic monoclonal antibody recognizes a consensus recognition site for phosphatidylserine in phosphatidylserine‐specific monoclonal antibody and protein kinase C
Authors: Reza Farooq , Igarashi Koji , Tokita Shigeru , Asai Kenji , Aoki Junken , Asaoka Yoshinori , Umeda Masato , Inoue Keizo
Published: July 2002

In order to elucidate the molecular mechanisms responsible for the specific lipid‐protein interactions, we have undertaken structural and idiotypic analyses of a monoclonal antibody, PS4A7, which binds specifically to phosphatidylserine (PS). Here we showed that one of the anti‐idiotypic monoclonal antibodies raised against PS4A7 cross‐reacted extensively with protein kinase C (PKC) and inhibited the activation of the enzymatic activity. The binding of the anti‐idiotypic antibody to PKC was inhibited specifically by PS, but not by other phospholipids including 1,2‐diacyl‐sn‐glycero ‐3‐phospho‐d‐serine or 1,2‐diacyl‐sn‐glycero‐3‐phospho‐l‐homoserine. In contrast, the binding of the anti‐idiotypic mAb to the enzyme was significantly enhanced in the presence of either diacylglycerol or sphingosine. These findings indicate that the PS‐specific monoclonal antibody and PKC share a consensus structure which is responsible for the specific interaction with PS and both diacylglycerol and sphingosine may induce a similar conformational change which exposes the PS‐specific binding site of the enzyme.