bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and Biochemical Characterization of the Trichoderma Reesei Hydrophobin HFBI
Authors: Nakari‐Setälä Tiina , Aro Nina , Kalkkinen Nisse , Alatalo Edward , Penttilä Merja
Published: August 2004

The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose‐containing medium, was shown to encode a novel fungal hydrophobin. The encoded 97‐amino‐acid protein is cysteine‐rich and has a typical signal sequence for secretion. Signal‐sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids. Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T. reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose‐containing cultures. The identity of HFBI was verified by N‐terminal and peptide sequencing or proteins purified both from the cell wall and culture medium. In the cell wall most of the HFBI formed SDS‐insoluble complexes that could be extracted with trifluoroacetic acid. Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus.
Amino Acid Preferences in the Octapeptide Subunit of the Major Histocompatibility Complex Class I Heterotrimer H‐2Ld
Authors: Pridzun Lutz , WiesmÜLler Karl‐Heinz , Kienle Stefan , Jung Günther , Walden Peter
Published: August 2004

Major histocompatibility complex class I (MHC‐I) molecules are heterotrimers composed of polymorphic α‐chains, monomorphic β‐chains, and peptides of eight or nine amino acids. The peptides are derived from various intracellularly occurring proteins and are very heterogeneous. They are essential for a stable conformation of the MHC‐I protein at physiological temperature. This study presents results from stabilization experiments that were designed to determine the impact of the amino acids in every sequence position of octapeptides on the thermal stability of the mouse MHC‐I molecule H2‐Ld. OX7 octapeptide libraries with one defined and seven randomized positions were employed as they allow the effects of individual amino acids to be determined. The results confirm the importance of the motif amino acids proline and leucine for positions 2 and 8, respectively, of octapeptides. They are among the most efficient amino acids for these positions. However, with a few exceptions, all amino acids are permitted in all eight sequence positions. Hydrophobic amino acids are generally favored. Charged amino acids, especially aspartic acid and glutamic acid, are disfavored. Stabilization indices were defined as measures for the MHC stabilization power of the amino acids. These indices can serve to predict the efficiency of peptide binding to H‐2Ld and can guide the design of T‐cell epitopes.
Nisin Z, Mutant Nisin Z and Lacticin 481 Interactions with Anionic Lipids Correlate with Antimicrobial Activity
Authors: Demel Rudolf A. , Peelen Tamara , Siezen Roland J. , De Kruijff Ben , Kuipers Oscar P.
Published: August 2004

Monomolecular layers of lipids at the air/water interface have been used as a model membrane to study membrane interactions of the lantibiotic nisin. The natural lantibiotics nisin A and nisin Z proved to have a high affinity for the anionic lipids phosphatidylglycerol and bis(phosphatidyl)glycerol (cardiolipin). The interaction with zwitterionic phopholipids or neutral lipids is very low at surface pressures higher than 32 mN/m. Nisin, nisin mutants and lacticin 481 show a remarkable correlation between antimicrobial activity and anionic lipid interaction. The results indicate that primarily the N‐terminal part (residues 1–22) penetrates into the lipid phase. Reduction of the flexibility at positions 20–21 has a negative effect on monolayer interaction and activity. The C‐terminal part is probably responsible for ionic interactions of nisin in monomeric or oligomeric form with anionic lipids. In mixtures of anionic and zwitterionic lipids maximal interactions are found at approximately 70 mol/100 mol anionic lipid. Gram‐positive bacteria, which form the main target for nisin, are characterized by a high content of anionic lipids in the membrane. Monolayers formed of lipid extracts of bacteria sensitive to nisin were more strongly penetrated than those of bacteria relatively insensitive to nisin.
Primary Structure of Cyclohydrolase (Mch) from Methanobacterium thermoautotrophicum (Strain Marburg) and Functional Expression of the mch Gene in Escherichia coli
Authors: Vaupel Martin , Dietz Heiko , Linder Dietmar , Thauer Rudolf K.
Published: August 2004

The gene mch encoding N5,N10‐methenyltetrahydromethanopterin cyclohydrolase (Mch) in Methanobacterium thermoautotrophicum (strain Marburg) was cloned and sequenced. The gene, 963 bp, was found to be located at the 3′ end of a 3.5‐kbp Bam HI fragment. Upstream of the mch gene two open reading frames were recognized, one encoding for a 25‐kDa protein with sequence similarity to deoxyuridylate hydroxymethylase and the other encoding for a 34.6‐kDa protein with sequence similarity to cobalamin‐independent methionine synthase (MetE). The N‐terminal amino acid sequence deduced for the deoxyuridylate hydroxymethylase was identical to that previously published for thymidylate synthase (Tys Y) from M. thermoautotrophicum. The 3′ end of the tys Y gene overlapped by 8 bp with the 5′ end of the mch gene. Despite this fact, the mch gene appeared to be transcribed monocistronically as evidenced by Northern blot analysis and primer‐extension experiments. The mch gene was overexpressed in Escherichia coli yielding an active enzyme of 37 kDa with a specific activity of 30 U/mg cell extract protein.
MUC1 Glycoforms in Breast Cancer
Authors: Hanisch Franz‐Georg , Stadie Tanja R. E. , Deutzmann Frank , Peter‐Katalinic Jasna
Published: August 2004

A highly immunogenic peptide motif within the tandem repeat domain of MUC1 mucin is assumed to be exposed during development of breast cancer due to altered O‐glycosylation. To elucidate the structural aspects of these changes, we have isolated and analysed the integrated or secretory MUC1 glycoforms from carcinoma cell lines or solid tumors and from human milk. The buoyant densities measured in CsCl gradients for MUC1 glycoforms from cancer cells revealed heterogeneity of the physicochemical species and a significant reduction of their carbohydrate contents compared to MUC1 from skim milk. Immunoreactivity patterns of MUC1 glycoforms from tumor or T47D cells exhibited a lack of fucosylated Lewis blood‐group‐related antigens and the appearance of core‐type antigen sialyl‐(NeuG1)‐TF, Galβ1–3(NeuGlα2–6)GalNAc. Structural chemistry of MUC1 oligosaccharides demonstrated that the cancer‐associated glycoforms carry mainly sialylated trisaccharides NeuAcα2–3Galβ1–3GalNAc or NeuAcα2–6(Galβ1–3)GalNAc, exhibit a concomitant decrease in the ratio of GlcNAc/GalNAc, a reduction or disappearance of l‐fucose, and a partial substitution of N ‐acetylneuraminic acid by the N‐glycolylated variant. On comparison to the secretory MUC1 in human milk, the glycoforms on human milk fat globule membranes showed apparently identical patterns of O‐linked oligosaccharides with a preponderance of neutral polylactosamino‐glycans. During serum‐free cultivation of T47D cells over 4 weeks, the expression of secretory MUC1 glycoforms was inconsistent based on the decreasing contents of sialic acid and on the concomitant increase of immunodetectable TF antigen.
The Regulation of Enzymes Involved in Chlorophyll Biosynthesis
Authors: Reinbothe Steffen , Reinbothe Christiane
Published: August 2004

All living organisms contain tetrapyrroles. In plants, chlorophyll (chlorophyll a plus chlorophyll b) is the most abundant and probably most important tetrapyrrole. It is involved in light absorption and energy transduction during photosynthesis. Chlorophyll is synthesized from the intact carbon skeleton of glutamate via the C5 pathway. This pathway takes place in the chloroplast. It is the aim of this review to summarize the current knowledge on the biochemistry and molecular biology of the C5‐pathway enzymes, their regulated expression in response to light, and the impact of chlorophyll biosynthesis on chloroplast development. Particular emphasis will be placed on the key regulatory steps of chlorophyll biosynthesis in higher plants, such as 5‐aminolevulinic acid formation, the production of Mg2+ ‐protoporphyrin IX, and light‐dependent protochlorophyllide reduction.
Interaction between Photosystem I and Flavodoxin from the Cyanobacterium Synechococcus sp. PCC 7002 as Revealed by Chemical Cross‐Linking
Authors: Mühlenhoff Ulrich , Zhao Jindong , Bryant Donald A.
Published: August 2004

The interaction between photosystem I (PS I) and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was investigated by covalent cross‐linking in the presence of a hydrophilic crosslinker, N ‐ethyl‐3‐(3‐diaminopropyl)carbodiimide. Under the experimental conditions employed, five distinct cross‐linking products of flavodoxin and PS I subunits are formed. Immunoblot analyses show that these species are the result of cross‐linking of flavodoxin to PsaC, PsaD, an unidentified low‐molecular‐mass PS I polypeptide, and a 15‐kDa subunit. The latter has been indirectly identified as the PsaF subunit. Analysis of the interaction of flavodoxin with PS I from a psaE mutant indicates that the PsaE subunit is required for correct complex formation between flavodoxin and PS I, although this subunit is not directly crosslinked to flavodoxin. In addition, the cross‐linking products of PsaD with PsaC and PsaL, and PsaE with PsaF, are observed. The covalent complex of flavodoxin and PS I is shown to be fully inhibited with respect to electron transfer to soluble flavodoxin, ferredoxin or ferredoxin:NADP+ oxidoreductase.
Muconolactone Isomerase of the 3‐Oxoadipate Pathway Catalyzes Dechlorination of 5‐Chloro‐Substituted Muconolactones
Authors: Prucha Matthias , Peterseim Anke , Timmis Kenneth Nigel , Pieper Dietmar Helmut
Published: August 2004

An enzyme of Alcaligenes eutrophus JMP 134 which catalyzes dechlorination of (4R, 5R)‐and (4R, 5S)‐5‐chloro‐3‐methyl‐and (4R, 5S)‐5‐chloromuconolactone to principally 3‐methyl‐trans‐, 3‐methyl‐cis ‐dienelactone and cis ‐dienelactone, respectively, was purified to homogeneity. The enzyme was identified as muconolactone isomerase on the basis of its high activity with muconolactone and on its high degree of sequence similarity with previously described muconolactone isomerases.

Molecular mass determinations of the highly hydrophobic and heat‐resistant enzyme indicated a decameric structure involving a single 10.100‐kDa subunit similar to that of muconolactone isomerase of Pseudomonas putida.

Kinetic analysis showed cooperative effects between the subunits during conversion of (4R, 5S)‐5‐chloro‐3‐methylmuconolactone. (4R, 5S)‐5‐chloromuconolactone was the preferred substrate, over the natural substrate (4S)‐muconolactone. The (4S, 5S)‐structure was found to be an inhibitor of (4R, 5R)‐5‐chloro‐3‐methylmuconolactone transformation. Methylsubstitution of the substrate results in a higher affinity for the enzyme, but a drastically lower velocity, resulting in a lower specificity constant.
hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System
Authors: Dernedde Jens , Eitinger Thomas , Patenge Nadja , Friedrich Bärbel
Published: August 2004

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD‐reducing cytoplasmic enzyme (SH) and a membrane‐bound electron‐transport‐coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545–553]. Re‐examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non‐polar in‐frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild‐type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2‐activating SH subunit (HoxH) at levels comparable to those observed in the wild‐type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild‐type strain, HoxH and the MBH large subunit (HoxG) undergo C‐terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni‐incorporation experiments identified both hydrogenases as nickel‐free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild‐type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.
Three‐Dimensional Structure of the Lantibiotic Nisin in the Presence of Membrane‐Mimetic Micelles of Dodecylphosphocholine and of Sodium Dodecylsulphate
Authors: Van Den Hooven Henno W. , Doeland Chantal C. M. , Van De Kamp Mart , Konings Ruud N. H. , Hilbers Cornelis W. , Van De Ven Frank J. M.
Published: August 2004

The lantibiotic nisin is a cationic, polycyclic bacteriocin of 34 residues, including several unusual dehydro residues and thioether‐bridged lanthionines. The primary target of its antimicrobial action is the cytoplasmic membrane. Therefore the conformation of nisin when bound to membrane‐mimicking micelles of zwitterionic dodecylphosphocholine and of anionic sodium dodecylsulphate was determined with high‐resolution NMR spectroscopy. Structures were calculated on the basis of NMR‐derived constraints with the distance‐geometry program DIANA and were further refined by restrained energy minimization using X‐PLOR. The conformation of nisin complexed to both types of micelles is the same, irrespective of the different polar head‐groups of the detergents. The structure consists of two structured domains: an N‐terminal domain (residues 3 – 19) containing three lanthionine rings, A, B and C; and a C‐terminal domain (residues 22–28) containing two intertwined lanthionine rings numbered D and E. These domains are flanked by regions showing structural variability. Both domains are clearly amphipathic, a property characteristic for membrane‐interacting polypeptides. The structures of the ring systems are better defined than those of the linear segments. The four‐residue rings B, D and E of nisin all show a β‐turn structure, which is closed by the thioether linkage. The backbones of the rings B and D form type I β‐turns. Ring E resembles a type II β‐turn. Preceding the intertwined rings D (residues 23–26) and E (25–28) another type41 β‐turn is found formed by the residues 21–24, so that the C‐terminal domain consists of three consecutive β‐turns. The structures of nisin in the micellar systems differ significantly from the previously determined (and now partially recalculated) structure in aqueous solution [van de Ven, F. J. M., van den Hooven, H. W., Konings, R. N. H. & Hilbers, C. W. (1991) Eur. J. Biochem. 202, 1181–11881 in the first lanthionine ring around dehydroalanine 5.
Hormonal Regulation of Aldose Reductase in Rat Ovary During the Estrous Cycle
Authors: Iwata Nobuhisa , Hara Shuichi , Nishimura Chihiro , Takahashi Makoto , Mukaii Toshiji , Takayama Masaomi , Endo Takahiko
Published: August 2004

The physiological roles of aldose reductase [alditol:NAD(P)+ 1‐oxidoreductase] have not been fully elucidated yet, although it has been implicated in the pathogenesis of diabetic complications. In the rat ovary we found remarkable changes in the enzyme level during the 4‐day estrous cycle. After diestrus, the activity and protein content of aldose reductase increased to the maximum level on proestrous morning and rapidly fell off to the lowest level on the early morning of estrus. At this time its mRNA level in the ovary was significantly decreased compared with that on the morning of proestrus. Immunohistochemical staining of the diestrous ovary demonstrated localization of the enzyme protein in the granulosa cells and in the oocytes. At the end of proestrus when its level was low, immunoreactive aldose reductase in the granulosa cells was localized preferentially to the antrum side, with lesser staining in the cells lining the follicles. Administration of chlorpromazine to the rats on proestrus significantly restored the enzyme level on the following morning of the expected estrus. This effect of chlorpromazine was abolished when human chorionic gonadotropin was administered to the chlorpromazine‐treated rats. When chlorpromazine was administered to the rats treated with bromocriptine, an inhibitor of pituitary prolactin secretion, aldose reductase activity in the ovary was significantly elevated compared with that in the rats treated with chlorpromazine alone. These findings suggest that in the rat ovary it is under hormonal regulation during the estrous cycle. The enzyme may possess a new functional role in the reproductive system of the female rat, which can be disordered under diabetic conditions.
Purification and cDNA Cloning of Ferritin from the Hepatopancreas of the Freshwater Crayfish Pacifastacus leniusculus
Authors: Huang Tien‐sheng , Law John H. , SöDerhäll Kenneth
Published: August 2004

Ferritin was purified from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus after injection of iron. It has the same size as horse spleen ferritin (440 kDa) and migrates as two bands, 19 kDa and 20 kDa, respectively, in SDSPAGE under reducing conditions. Crayfish ferritin (20 kDa) was cloned from a hepatopancreas cDNA library. The deduced amino acid sequence of the crayfish ferritin shows a closer relationship to vertebrate ferritin heavy chains than to insect ferritin and contains the conserved H‐specific residues for the ferroxidase centre found in vertebrate ferritin heavy chain. An IRE(iron‐responsive element)‐like sequence with a predicted stem‐loop structure was present in the 5′ untranslated region of the crayfish ferritin mRNA. Crayfish ferritin does not share the atypical properties of insect ferritins, such as high molecular mass of intact protein, abundance in hemolymph, and export into vacuoles. We suggest that there are two different types of ferritins distributed in different species: insect‐type or secretory ferritins which are predominant in the snail oocyte and insects, and vertebrate (crustacean)‐type or cytosolic ferritins which are predominant in vertebrates and crustacea.
Oxidation of Dimethoxylated Aromatic Compounds by Lignin Peroxidase from Phanerochaete Chrysosporium
Authors: Joshi Dinesh K. , Gold Michael H.
Published: August 2004

The stabilities of the cation radicals of veratryl alcohol, 3,4‐dimethoxytoluene and 1,4‐dimethoxy‐benzene were compared by monitoring the formation of dimeric products during the oxidation of these substrates by lignin peroxidase (LiP). LiP oxidized veratryl alcohol to generate veratraldehyde as the major product. Several other monomeric products were obtained in low yield. Dimeric products resulting from the coupling of two cation radicals, or a cation radical with a neutral molecule, were obtained only in trace amounts or not at all. This suggests that the cation radical of veratryl alcohol rapidly loses a benzylic proton to form a benzylic radical which undergoes further reactions to form veratraldehyde. In contrast, the LiP oxidation of 3,4‐dimethoxytoluene generated the dimeric product 3‐(2,3‐dimethoxy‐6‐methylphenyl)‐4‐methyl‐l,2‐benzoquinone as the major product. Several other monomeric and dimeric products were produced in lower yields. The generation of these dimeric products indicates that the cation radical of 3,4‐dimethoxytoluene is considerably more stable than that of veratryl alcohol. This suggests that the electronegative benzylic oxygen of veratryl alcohol increases the acidity of the benzylic protons, destabilizing the veratryl alcohol cation radical. LiP oxidized 1,4‐dimethoxybenzene to generate 1,4‐benzoquinone and 2‐(2,5‐dimethoxyphenyl)‐l,4‐benzoquinone as the major products. The formation of these products indicates that the cation radical of 1,4‐dimethoxybenzene also is relatively stable, as previously demonstrated by ESR. All of these results indicate that the veratryl alcohol cation radical generated by LiP oxidation is unstable, suggesting that it would not act as a diffusible radical mediator in LiP‐catalyzed reactions.
cDNA Cloning of a Novel Protein Containing two zinc‐finger Domains that may Function as a Transcription Factor for the Human Heme‐oxygenase‐1 Gene
Authors: Muraosa Yasuhi , Takahashi Kazuhiro , Yoshizawa Miki , Shibahara Shigeki
Published: August 2004

Heme oxygenase 1 is an essential enzyme in enzyme in heme catabolism that cleaves heme to form biliverdin, iron, and carbon monoxide. The human heme‐oxygenase‐1 gene is transcriptionally activated through the cis ‐regulatory clement (MTE), GTCATATGAC (positions −156 to −147), during 12‐O ‐telradccnnoyl‐phorbol 13‐acetate (TPA)‐induced differentiation of myelomonoeytic cell lines; such as THP‐1, to macro‐phages. MTE is responsible for the myelomonocytic‐specific induction of heme‐oxygenitsc‐1 gene expression, and is bound by ubiquitous and myelomonocytic cell‐line‐restricted proteins. In this study, we cloned the cDNA segments coding for a portion of a protein that binds to MTE by a Southwestern procedure from a THP‐1 cDNA expression library; we subsequently isolated putative full‐length cDNAs by a conventional hybridization procedure. The deduced protein, trended MTB‐Zf, consists of 1482 amino acid residues, has a molecular mass of about 162 kDa, and contains the two widely separated zinc‐finger domains located near the N‐ and C‐terminal. MTR‐Zf possesses other structural features characteristic of transcription factors, including a long stretch of acidic amino acids (amino acids 67–95), a proline‐rich region (positions 733–8491, a region rich in basic amino acids (positions 1161–1247), and a leueinc repeat‐like region (positions 486–514). We show that a portion of MTB‐Zf, including an N‐terminal zinc‐finger domain, binds in vitro to MTE and that the transient coexpression of MTB‐Zf cDNA leads to transativation of the heme‐oxygenase‐1 gene promoter. Since the 6.5‐kb MTB‐Zf mRNA is expressed in various human cell lines of different lineages, MTB‐Zf may represent a ubiquitous MTE‐binding protein. Furthermore, the MTB‐Zf gene has been in mapped to human chromosome 1p35–36.1 by tluorescence in situ hybridization, a region which is frequently deleted in various solid tumors, including neuropenic tumors. We found remarkable differences in the expression patterns of MTB‐Zf mRNA and two other hybridizable mRNAs of 5kb and 8.5 kb when human brain and primary brain tumors were compared. Both MTB‐Zf and the 8.5‐kb mRNAs were abundantly expressed in the five primary brain tumors examined, but only the 5‐kb mRNA was detectable in the human brain. These results suggest that MTB‐Zf is a transcription factor and may also play an important role in cell growth or differentiation
Overexpresson of RIIβ, Regulatory Subunit of Protein Kinase A in Human Colon Carcinoma Cell Induces Growth Arrest and Phenotypic Changes that are Abolished by Site‐Directed Mutation of RIIβ
Authors: Nesterova Maria , Yokozakt Hiroshi , McDuffie Elwood , Cho‐Chung Yoon S
Published: August 2004

LS‐174T human colon carcinoma cells that contain approximately equal amounts of cAMP‐dependent protein kinase (PKA) isozymes, PKA‐I and PKA‐II, were infected with retroviral vectors coding for regulatory (R) and catalytic (C) subunits of human PKA. In cells overexpressing RIIα, RIIβ, and RIIβ‐P (a RIIβ, mutant at the autophosphorylation site), PKA‐II levels increased whilc PKA‐I levels decreased. PKA‐I was almost completely eliminated in cells over expressing RIIβ or RIIβ‐P. In contrast, over expression of either RIα, or Cα had little or no effect on PKA isozyme levels. Although all infectants expressed high levels of PKA subunit mRNAs in accordance with gene introduction, the R subunit protein expression was reflected in PKA isozyme levels rather than in subunit mRNA levels. Only RIIβ infectants demonstrated marked growth inhibition in monolayer culture, reduced thymidine incorporation into DNA, and inability to grow in semisolid medium or in serum‐free medium. Conversely, all other infectants displayed growth properties similar to uninfected parental cells. The growth‐retardation properties of RIT, infetants were reflected in their altered phenotypic appearances. Our findings that the mutant RIIβ P could not mimic the growth‐inhibitory effect of RIIβ P suggest the functional importance of the autophosphurylation site in RIIβ, Our results suggest a role for RIIβ in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may be involved in neoplastic transformation.
Isolation and Characterization of the Major Gel Proteins in Human Semen, Semenogen I and Semenogen II
Authors: Malm Johan , Hellman Jukka , Magnusson Helena , Laurell Carl‐Bertil , Lilja Hans
Published: August 2004

Semenogen I and semenogen II constitute the major gel‐forming proteins in human semen. The gel proteins were rapidly solubized and separated from spermatozoa in ejaculates collected at pH 9.7 in buffer containing 4 mol/1 urea and dithiothreitol. This protected the semenogens from proteolytic degradation by prostate‐specific antigen, and allowed their isolation by affinity chromatography on heparin‐Sepharose. Semenogens I and II were almost selectively retained and eluted partially separated in 0.25 mol/1 NaCl. Further purification was achieved by chromatography on Superose. Approximately 10–20 mg semenogen I and 2–5 mg semenogen II were recovered from each sample with a purity exceeding 95% as judged by SDS/PAGE. The molecular mass of semenogen I (49 958 Da) and the major form of semenogen II (63539 Da) measured by mass spectrometry was consistent with the reported cDNA data. The occurrence of a second, larger form of semenogen II was due to asparagine‐nked glycosylation. The amino‐termini of the purified proteins were blocked, but digestion with pyroglutamate amino‐peptidase enabled the identification of amino‐terminal sequences consistent with the reported cDNA data. The amino acid compositions of the purified proteins were also consistent with those derived from cDNA data. The absorption coefficients (280 nm, 1%, 1 cm) for semenogens I and II were 5.5 and 5.4, respectively, and the isoelectric point was above pH 9.5 for both proteins.
Molecular Cloning and Functional Expression of the α1A‐adrenoceptor of Medaka Fish, Oryzias latipes
Authors: Yasuoka Akihito , Abe Keiko , Arai Soichi , Emori Yasufumi
Published: August 2004

A genomic DNA encoding a subtype adrenoceptor (AR) was cloned from Medaka fish, Oryzias latipes, using an oligonucleotide probe corresponding to the consensus sequence of mammalian α‐AR and β‐AR. The gene spans at least 9 kbp, and the coding region consists of two exons split by an intron of 7.2 kbp located at the same position as those of mammalian α1B‐AR genes. The gene encodes 470 amino acid residues, the sequence of which shows the highest similarity to that of mammalian α1A‐AR (61%) and significant but lower similarities to other α‐AR and β‐AR proteins (31–45%), indicating that the gene encodes a Medaka homolog of α1A‐AR. To characterize the encoded protein, the mRNA was synthesized in vitro and injected into Xenopus oocytes. As a result, the oocytes responded lo 100 nM epinephrine evoking a Ca2+‐dependent Cl− current in the order of microamperes, which was not observed for oocytes injected with water alone. The response was reversibly inhibited by α1‐selective antagonist, WB4101 (2‐([2,6‐dimethoxyphenoxyethyl]aminomethyl)‐1,4‐benzodioxane). Similar experiments using several adrenergic agonists revealed that Medaka α1A‐AR responds to the following agonists in the order: epinephrine 2 ≥(−)norepinephrine > oxymetazoline ≥ methoxamine, which is similar to the responses of rat α1A receptor expressed in COS cells. The results indicate that fish contains adrenoceptor systems similar to those of mammals in terms of primary structure and pharmacological properties.
A Tobacco Nuclear Protein that Preferentially Binds to Unmethylated CpG‐rich DNA
Authors: Fisscher Ursula , Weisbeek Peter , Smeekens Sjef
Published: August 2004

The methylation of cytosine residues in CpG dinucleotides of eukaryotic DNA is an important mechanism for the regulation of gene expression. Higher plants have a high content of methylated cytosine residues in CpG as well as CpNpG sites, and experimental evidence suggests a role in gene expression for DNA methylation. In this article, we describe a tobacco nuclear protein whose binding to various DNA sequences is positively correlated with the CpG density of the probes. This protein, CpG‐binding protein 1 (CGBP‐1), has reduced affinity for DNA when the CpG sites are methylated. Ribonuclease treatment also reduces the formation of the CGBP‐1 complex. The binding characteristics of CGBP‐1 make it an interesting protein with respect to methylation‐mediated gene expression in p1 nts.
Transcriptional Regulation of Transferrin Receptor Expression During Phorbol‐Ester‐Induced HL‐60 Cell Differentiation
Authors: Lok Chun Nam , Chan Kai‐Foon Jesse , Loh Tatt Tuck
Published: August 2004

The mechanism involved in the regulation of transferrin receptor (TfR) expression during phorbol‐ester‐induced HL‐60 cell differentiation was investigated. The mRNA of the TfR was constitutively expressed in proliferating HL‐60 cells. Treatment of the cells with phorbol 12‐myristate 13‐acetate (PMA) for 24 h resulted in a gradual decrease in the expression of the TfR mRNA. Nuclear run‐on assays revealed that the transcription of the TfR gene was inhibited by prior treatment of cells with PMA. The effect of PMA on the binding of nuclear proteins to the TfR gene promoter region was then investigated. Based on sequence similarity and previous footprinting data, the promoter region of the TfR gene seems to contain a sequence like that of the phorbol‐ester‐responsive element (TRE). Our results showed that the binding of nuclear extracts to the TfR gene promoter region containing the TRE‐like sequence was increased in PMA‐treated cells. This binding activity could be abolished by prior incubation of the nuclear extracts with a synthetic oligonucleotide containing the consensus TRE sequence. In vitro transcription assays revealed that prior incubation of the nuclear extracts of PMA‐treated cells with excess consensus TRE oligonucleotide enhanced the gene transcription driven by the TfR gene promoter. These findings suggest that the TRE‐like element may play a role in the inhibition of TfR gene transcription.
A 13C–NMR Study of the Mechanism of Bacterial Metabolism of Monomethyl Sulfate
Authors: Higgins Timothy P. , Hewlins Michael J. E. , White Graham F.
Published: August 2004

Two different mechanisms have been proposed previously for initiating the biodegradation of monomethyl sulfate (MeSO4) in bacteria. For a Hyphomicrobium species, a sulfatase enzyme has been proposed to hydrolyse MeSO4 to methanol and inorganic sulfate. For an Agrobacterium sp., an alternative proposal involves monooxygenation of MeSO4 (hydroxylation) to produce methanediol monosulfate, which decomposes spontaneously to formaldehyde and inorganic sulfate. In the present study, 13C‐NMR was used to monitor metabolic intermediates of [13]MeSO4 in real time in each species in order to resolve the issue of mechanism of biodegradation. Agrobacterium sp. M3C grew on MeSO4 but not on methanol. MeSO4‐grown cells catabolised [13C]MeSO4 but not [13C]methanol, and [13C]methanol did not accumulate from MeSO4 in the presence of a known inhibitor or methanol dehydrogenase (cyclopropanol). Hyphomicrobium MS223 grew on MeSO4 and, in contrast with the Agrobacterium sp., also on methanol. The normally rapid metabolism of [13C] methanol by methanol‐grown cells was arrested by cyclopropanol, but metabolism of [13C]MeSO4 by MeSO4‐grown cells was unaffected. Moreover there was no accumulation of [13C] methanol from [13C]MeSO4 under conditions in which methanol dehydrogenase was shown to be inactive. The results provided strong evidence against the intermediacy of methanol in the biodegradation of MeSO4 in either species, and thereby render untenable mechanisms involving sulfatase‐mediated hydrolysis of MeSO4. The data are consistent with the hydroxylation of MeSO4 via a monooxygenation mechanism and subsequent spontaneous hydrolysis of the methanediol monosulfate intermediate.
1H‐NMR Signal of Arenicola marina Myoglobin in vivo as an Index of Tissue Oxygenation
Authors: Kreutzer Ulrike , Jue Thomas
Published: August 2004

Key questions on how intertidal animals adapt to hypoxic stress center on the high energy phosphate response to decreasing oxygenation. With recent 1HP/31P‐NMR techniques to monitor mammalian tissue metabolism, a novel approach has emerged to observe potentially the intracellular oxygen interaction in invertebrates. The present study indicates that Arenicola marina, a standard model for intertidal animals, exhibits a distinct set of Mb 1H‐NMR signals in vivo, corresponding to the two isolated Mb isoforms. Specitically both deoxy‐Mb I and deoxy‐Mb IT exhibit paramagnetically shifted signals at 93.4 ppm and 92.5 ppm at 25 °C, respectively, which arise from the proximal histidyl NδH. These signals reflect the cellular oxygenation state and indicate clearly that the phasphotautocyamine level begins to drop at the onset of anoxia and declines gradually to 50% of control after 3.5 h. 1H Mb spectra indicate protein heterogeneity originating from heme as well as structural disorder.
Changes in Plasma Phospholipids in the Presence and Absence of Erythrocytes
Authors: Nourt‐Sorkhabi M. Hossein , Sullivan David R. , Benton Christopher M. , Kuchel Philip W.
Published: August 2004

Whole human blood and plasma were incubated at 37°C and 31P‐NMR spectra were acquired from aliquots of plasma dissolved in sodium cholate. The narrow resonances of phospholipids were well resolved, allowing identification and accurate quantification of the various phospholipid classes. During the course of plasma incubation, the rate of increase in 2‐lysophosphatidylcholine (2‐LysoPtdCho) corresponded closely to the rate or decrease in phosphatidylcholine. However, little Or no change was observed in the sphingomyelin concentrations. The rate of cholesteryl ester formation in plasma was also determined using HPLC and was observed to be similar to the rate of 2‐LysoPtdCho increase in plasma; this is consistent with the rate of acyl transfer from phosphatidylcholine to free cholesterol catalysed by lecithin:cholesterol acyltransferase. However, during the course of whole blood incubation, the rate of 2‐LysoPtdCho increase in plasma was significantly lower than the rate of 2‐LysoPtdCho production during incubation of plasma alone (31±4 μmol · 1−1· h−1 and 80±8 μmol · 1−1· h−1 respectively). The difference between the rates can be attributed to the action of enzymes present in blood cells including those in the erythrocytes that catalyse acylation or hydrolysis of 2‐LysoPtdCho.
T‐cell Membrane‐Associated Serine Protease, Tryptase TL2, Binds Human Immunodeficiency Virus Type 1 gp120 and Cleaves the Third‐Variable‐Domain Loop of gp120
Authors: Niwa Yasuharu , Yano Mihiro , Futaki Shiroh , Okumura Yuushi , Kido Hiroshi
Published: August 2004

It has been suggested that the third variable domain (V3) loop of human immunodeficiency virus type 1 (HIV‐1) gp120 has to interact with a cell‐surface‐associated protease(s) that acts as a cofactor after binding of gp120 to the CD4 receptor during entry of HIV‐1 into susceptible cells. We isolated the membrane‐associated serine protease, tryptase TL2, from human CD4‐positive lymphocytes. This enzyme specifically binds gp120 through interaction with its V3 domain. To investigate the role of tryptase TL2 in HIV infection, we examined the affinity of the interaction and the proteolytic susceptibility of various recombinant gp120 expressed in mammalian cells to the enzyme, and we determined the cleavage sites. Tryptase TL2 bound gp120 with an apparent dissociation constant of 38 nM. The affinity was lower than that of gp120 for CD4 which suggests that gp120 initially binds to CD4, followed by interaction with tryptase TL2 which is localized close to CD4 on the cell surface. After binding, tryptase TL2 cleaved recombinant gp120 expressed in mammalian cells into two protein species of 70 kDa and 50 kDa but did not cleave gp120 expressed in insect cells, which indicates that the structure of the oligosaccharides linked to the polypeptide backbone of gp120 affects the proteolytic susceptibility. Cleavage was specifically inhibited by a neutralizing antibody against the V3 loop. Cleavage‐site determination revealed that tryptase TL2 cleaved gp120 at various sites in the V3 in a strain‐dependent manner. The amino acid variability at the cleavage site(s) in almost all HIV‐1 isolates was restricted to amino acids which are susceptible to the chymotryptic and/or tryptic activities of tryptase TL2.
The CO Stretching Mode Infrared Spectrum of Substrate‐Free Cytochrome P ‐450cam—CO
Authors: Jung Christiane , Ristau Otto , Schulze Heike , Sligar Stephen G.
Published: August 2004

The effect of pH, glycerol, temperature, and pressure on the carbon monoxide (CO) stretch mode of substrate‐free cytochrome P‐450cam (CYP101) was studied. Complex spectra of overlapping bands have been observed. CO stretch bands centered at about 1911–1918 cm 1(band I), 1927–1931 cm−1 (band II), 1940–1942 cm−1 (band III), 2950–1953 cm−1 (band IV), 1960–1963 cm−1 (band V) and 1966–1973 cm−1 (band VI) are obtained from the fitting analyses independently of the lineshape model used. Only two or three bands are dominant in each spectrum. Compared to bands I, II and III, the bands IV and V are assigned to correspond to a weaker polar contact between the CO ligand and a polar group in the heme pocket (probably Thr252) because of the opposite effect of glycerol (osmotic pressure) and hydrostatic pressure on the intensity and frequency of these bands. The different CO stretch bands are interpreted as indicating conformational substates of the protein. It is suggested that water in the heme pocket plays an important role for the substate equilibrium. This substate equilibrium freezes in at the glass transition temperature, Tg the protein/solvent mixture. For the temperature region above Tgthe thermodynamic parameters and volumes for the substates have been determined by a global fit analysis of the temperature‐ and pressure‐dependent populations and they are compared to the respective values for camphor‐bound cytochrome P‐450cam.
Cloning of Arabidopsis thaliana Glutathione Synthetase (GSH2) by Functional Complementation of a Yeast Gsh2 Mutant
Authors: Ullmann Pascaline , Gondet Laurence , Potier Serge , Bach Thomas J.
Published: August 2004

Glutathione (L‐γ‐glutamyl‐L‐cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Arabidopsis thaliana was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Saccharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced M1: 53783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharomyces pombe). A highly conserved glycine‐rich domain close to the carboxy‐terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The M, is similar to those of soluble animal enzymes, suggesting that the Arabidopsis gene also codes for a cytosolic protein. Genomic DNA‐blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectibility in mutants to about 50% of the wild‐type levels in transformed cells.
Stereochemical Course and Reaction Products of the Action of β‐Xylosidase from Thermoanaerobacterium saccharolyticum Strain B6A‐RI
Authors: Armand Sylvie , Vieille Claire , Gey Claude , Heyraud Alain , Zeikus J. Gregory , Henrissat Bernard
Published: August 2004

β‐Xylosidases are grouped in families 39 and 43 of a general classification of glycosyl hydrolases based on amino acid sequence similarities [Henrissat, B. & Bairoch, A. (1993) Biochem. J. 293, 781–788]. The β‐xylosidase from Butyrivibrio fibrisolvens, which belongs to family 43, has been shown to operate by a molecular mechanism which results in the inversion of the anomeric configuration [Braun, C., Meinke, A., Ziser, L. & Withers, S. G. (1994) Anal. Biochem. 212, 259–262], Thermoanaerobacterium saccharolyticum B6A‐RI β‐xylosidase which belongs to family 39 was purified as a recombinant enzyme from Escherichia coli. The stereochemistry of the hydrolysis of p ‐nitrophenyl β‐d‐xylopyranoside was followed by 1H NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm (J≈ 10 Hz) assignable to H1 of free β‐xylose with a small amount of α‐xylose (5.05 ppm, J≈ 3 Hz) attributable to mutarotation. This result indicates that T. saccharolyticumβ‐xylosidase operates with overall retention of the anomeric configuration. This result, with the lack of sequence similarity between the two families of β‐xylosidases, suggests that these two families have major differences in their active‐site geometries. Consistent with its retaining mechanism, β‐xylosidase of T. saccharolyticum B6A‐RI also displayed transglycosylating activity: reverse‐phase HPLC showed approximately 30% conversion of p ‐nitrophenyl β‐D‐xylopyranoside into a number of higher nitrophenyl oligosaccharides after 5 min incubation with the enzyme. The structure of the most abundant oligosaccharides could be determined by total correlation spectroscopy NMR and showed that the enzyme can build β‐1,4, β‐1,3‐ and β‐1,2‐linked xylo‐oligosaccharides.
Probing the Aromatic‐Donor‐Binding Site of Horseradish Peroxidase Using Site‐Directed Mutagenesis and the Suicide Substrate Phenylhydrazine
Authors: Gilfoyle David J. , Rodriguez‐Lopez Jose Neptuno , Smith Andrew T.
Published: August 2004

The haem groups from two classes of site‐directed mutants of horseradish peroxidase isoenzyme C (HRP‐C) (distal haem pocket mutants, [H42L]HRP‐C* and [R38K]‐HRP‐C* and peripheral‐haem‐access‐channel mutants, [F142A]HRP‐C* and [F143A]HRP‐C*) were extracted and analysed by reverse‐phase HPLC after phenylhydrazine‐induced suicide inactivation. The relative abundance of the two covalently modified haems, C20‐phenyl (δ‐meso phenyl) and C18‐hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate‐access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP‐C* exhibited only approximately 0.03 % of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18‐methyl position although the level of arylation at the haem C20 position remained remarkably constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP‐C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP‐C wild‐type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic‐donor binding. We suggest reasons why phenylhydrazine‐treated C. cinereus peroxidase was more resistant to haem modification and phenyl‐radical‐based inactivation than HRP‐C.
Stereospecific Induction of Apoptosis in U937 Cells by N‐Octanoyl‐Sphingosine Stereoisomers and N‐Octyl‐Sphingosine
Authors: Karasavvas Nicos , Erukulla Ravi Kumar , Bittman Robert , Lockshin Richard , Zakeri Zahra
Published: August 2004

We investigated the ability of N‐octanoyl‐sphingosine (C8‐Cer) stereoisomers, N‐octanoyl‐DL‐erythro ‐dihydrosphingosine (DL‐e ‐DHC8‐Cer), and a new ceramide derivative, N‐octyl‐D‐erythro ‐sphingosine (D‐e ‐C8‐Ceramine), to induce apoptosis in U937 cells. We found the C8‐Cer stereoisomers to be stereo‐specific with the D‐ and L‐threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D‐t ‐C8‐Cer = L‐t ‐C8‐Cer > L‐e ‐C8‐Cer > D‐e ‐C8‐Cer > DL‐e ‐DHC8‐Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D‐e ‐C8‐Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D‐e ‐C8‐Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide‐activated protein kinase (CAPK). While the potent DNA fragmentation‐inducing compounds D‐e ‐C8‐Ceramine and L‐t ‐C8‐Cer failed to increase the cellular ceramide levels, D‐e ‐C8‐Cer, D‐t ‐C8‐Cer and D‐e ‐C8‐Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation‐inducing ability of the threo stereoisomers and D‐e ‐C8‐Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D‐e ‐C8‐Ceramine and L‐t ‐C8‐Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D‐e ‐C8‐Cer.