bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterization of the phosphatidylinositol‐3,4,5‐trisphosphate Phosphatase in Bovine Thymus
Authors: Kabuyama Yukihito , Nakatsu Noriyuki , Homma Yoshimi , Fukui Yasuhisa
Published: August 2004

Using phosphatidylinositol 3,4,5‐trisphosphate [PtdIns(3,4,5)P3] prepared from phosphatidylinositol 4,5‐bisphosphate and inositolphospholipid 3‐kinase, we identified in bovine thymus extracts the enzyme activity which catalyzed dephosphorylation of PtdIns(3,4,5)P3, to produce phosphatidylinositol bisphosphate. Since bovine thymus exhibited the highest level of activity among tissues screened, we tried to purify this enzyme PtdIns(3,4,5)P3 phosphatase from bovine thymus. After sequential chromatographies using S‐Sepharose, heparin‐Sepharose, blue Sepharose, and Toyopearl HW55, the enzyme was purified 1875‐fold with a yield of 10%. SDS/PAGE analysis revealed that a 120‐kDa protein band copurified with the enzyme activity. The apparent molecular mass of the active protein was 120 kDa on size‐exclusion chromatography, suggesting that the 120‐kDa band on SDS/PAGE is the PtdIns(3,4,5)P3 phosphatase. Since PtdIns(3,4,5)P3 phosphatase seemed to be the only activity that metabolized PtdIns(3,4,5)P3, and the enzyme did not hydrolyze phosphatidylinositol 4,5‐bisphosphate, the enzyme may play a critical role in the inositolphospholipid 3‐kinase signalling.
Two Additional 5′ Exons in the Human Vigilin Gene Distinguish it from the Chicken Gene and Provide the Structural Basis for Differential Routes of Gene Expression
Authors: Kügler Sebastian , Plenz Gabriele , Müller Peter K.
Published: August 2004

Vigilin, a 150‐kDa protein, contains 14 tandemly arranged domains, each consisting of a KH RNA‐binding motif and a spacer region. Here, we report on the physical structure of the human Vigilin gene with 29 exons, thereby outnumbering the chicken gene by two additional 5′ exons. These additional exons, 1A and 1B, are alternatively though concurrently spliced to exon 1C which is homologous to the first exon in the chicken gene. None of the additional human exons code for an amino‐terminal extension of Vigilin, due to in‐frame stop codons. Structural features of exon 1A, however, would allow the translation of a 13‐amino‐acid peptide from an upstream open reading frame preceding the vigilin open reading frame. We suggest that exons 1A and 1B have been gained during evolution, allowing alternative routes of expression control of the human Vigilin gene.
NMR Detection of Arginine‐Ligand Interactions in Complexes of Lactobacillus casei Dihydrofolate Reductase
Authors: Gargaro Angelo R. , Frenkiel Thomas A. , Nieto Pedro M. , Birdsall Berry , Polshakov Vladimir I. , Morgan William D. , Feeney James
Published: August 2004

1H‐NMR and 15N‐NMR signal assignments have been made for the eight arginine residues in Lactobacillus casei dihydrofolate reductase in its binary complex with methotrexate and in its ternary complex with methotrexate and NADPH. 1H‐NMR chemical shifts for the guanidino groups of two of the arginines (Arg57 and Arg43) were sensitive to different modes of binding of the guanidino groups with charged oxygen atoms of the ligands. In the complexes formed with methotrexate, Arg57 showed four non‐equivalent NHη proton signals indicating hindered rotation about the Nɛ‐Cζ and Cζ‐Nη bonds. The NHη12 and NHη22 protons showed large downfield shifts, which would be expected for a symmetric end‐on interaction of these protons with the charged oxygen atoms of a carboxylate group in methotrexate. These effects were not observed for the complex formed with trimethoprim, which does not contain any carboxylate groups. In the complex formed with NADPH present, Arg43 showed a large downfield chemical shift for its NHɛ proton and a retardation of its rate of exchange with water. This pattern of deshielding contrasts with that detected for Arg57 and is that expected for a side‐on interaction of the guanidino group protons with charged oxygen atoms of the ribose 2′‐phosphate group of NADPH.
Phosphorylation of Proteasomes in Mammalian Cells
Authors: Mason Grant G. F. , Hendil Klavs B. , Rivett A. Jennifer
Published: August 2004

The proteasome, a multimeric protease, plays an important role in nonlysosomal pathways of intracellular protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are phosphorylated and to investigate the possible role of phosphorylation in regulating proteasome activity and the association with regulatory components. Rat‐1 fibroblasts were grown in the presence of [32P]phosphate and proteasomes were immunoprecipitated from cell lysates with proteasome‐specific polyclonal antibodies. Subsequent analysis by two‐dimensional polyacrylamide gel electrophoresis showed two radiolabeled proteasome subunits which were identified using monoclonal antibodies as C8 and C9. Treatment of human embryonic lung cells (L‐132), under identical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in both C8 and C9. Examination of the sequence of C9 showed a potential cGMP‐dependent phosphorylation site (‐Arg3‐Arg‐Tyr‐Asp‐Ser‐Arg8‐), whilst C8 contains several potential casein kinase II phosphorylation sites. Following immunoprecipitation by a monoclonal antibody and dephosphorylation by acid phosphatase, proteasomes were observed to have significantly lower activities when compared to phosphorylated proteasomes, implying that phosphorylation may be an important mechanism of regulating proteasome function. Free proteasomes were separated by gel‐filtration from those complexed with regulatory complexes to form the 26S proteinase. The ratio of phosphorylation of C8 and C9 was found to be very similar in the two complexes but the level of phosphorylation was higher in the 26S proteinase than in free proteasomes.
A New Method for the Determination of Specific13C Enrichment in Phosphorylated [1–13C]glucose Metabolites
Authors: Lutz Norbert W. , Yahi Nouara , Fantini Jacques , Cozzone Patrick J.
Published: August 2004

A31P‐NMR method for the determination of13C enrichment in phosphorylated [1‐13C]glucose metabolites was developed by taking advantage of the13C satellites detectable for31P‐NMR signals of metabolites such as UDP‐hexoses, UDP‐N‐acetylhexosamines and other phosphorylated compounds generated during glycolysis and subsequent anabolism. HT‐29 cells were incubated in culture medium containing 4.5 g/l [1‐13C]glucose for 24 h prior to cell extraction, and high‐resolution31P‐NMR spectra were acquired from perchloric acid extracts. Since glucose and its phosphorylated products are key metabolites for many different metabolic processes, this method may be very helpful for studying specific metabolic pathways involving phosphorylated glucose metabolites.
Characterization and Molecular Cloning of Sambucus nigra Agglutinin V (Nigrin b), A Galnac‐specific Type‐2 Ribosome‐Inactivating Protein from the Bark of Elderberry (Sambucus nigra)
Authors: van Damme Els J. M. , Barre Annick , Rougé Pierre , van Leuven Fred , Peumans Willy J.
Published: August 2004

The molecular structure of the Sambucus nigra agglutinin V (SNAV), which has been described previously as a type‐2 ribosome‐inactivating protein called nigrin b, has been studied in detail by analysis of the purified protein combined with cDNA cloning and molecular modelling. Native SNAV is a dimer of two [A‐s‐s‐B] pairs. Hapten inhibition assays indicated that GalNAc is a 20‐fold more potent inhibitor of SNAV than Gal. A cDNA clone encoding SNAV was isolated from a cDNA library constructed with mRNA from the bark. Sequence analysis of this cDNA revealed a striking similarity to the recently cloned NeuAc α‐2,6‐gal/GalNAc‐specific S. nigra bark agglutinin I (SNAI) and to the previously sequenced type‐2 ribosome‐inactivating proteins from Ricinus communis and Abrus precatorius. In addition, molecular modelling of SNAV further suggested that its structure closely resembles that of ricin. The N‐terminal sequence of the B chain of SNAV also shows a marked similarity with the polypeptide of the previously described GalNAc‐specific S. nigra bark agglutinin II (SNAII), which unlike SNAV and SNAI has no ribosome‐inactivating activity. It appears, therefore, that elderberry bark contains at least two different type‐2 ribosome‐inativating proteins and a lectin built up of subunits which are closely related to the B chain of SNAV.
Recombinant Prespore‐Specific Antigen from Dictyostelium discoideum is a β‐sheet Glycoprotein with a Spacer Peptide Modified by O‐linked N‐acetylglucosamine
Authors: Zachara Natasha E. , Packer Nicolle H. , Temple Mark D. , Slade Martin B. , Jardine Daniel R. , Karuso Peter , Moss Catherine J. , Mabbutt Bridget C. , Curmi Paul M. G. , Williams Keith L. , Gooley Andrew A.
Published: August 2004

Prespore‐specific antigen (PsA) is a putative cell‐adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell‐surface proteins. It has an N‐terminal globular domain presented to the extracellular environment on an O‐glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl‐PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell‐surface molecules and here we present information on the structure of the N‐terminal globular domain and determine the reducing‐terminal linkage of the O‐glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H‐NMR spectra of rPsA contained features consistent with a high degree of β‐sheet in the N‐terminal globular domain, a feature commonly observed in cell‐adhesion proteins. Solid‐phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O‐glycosylations were determined to be single N ‐acetylglucosamine residues.
Assignment of 13C Resonances and Analysis of Relaxation Properties and Internal Dynamics of Pike Parvalbumin by 13C‐NMR at Natural Abundance
Authors: Alattia Temim , Padilla André , Cavé Adrien
Published: August 2004

Pike parvalbumin is an 11.5‐kDa globular protein which binds Ca2+ through EF‐hand structural motifs. Nearly complete assignment of the protonated 13C resonances has been achieved by means of hetero‐nuclear two‐dimensional experiments. The study shows that 13Cα chemical shifts can be very sensitive to localised conformational aspects. To characterise internal dynamics of pike parvalbumin, longitudinal‐relaxation and transverse‐relaxation rates and 1H‐13C NOEs were measured for α‐carbons at natural abundance by means of two‐dimensional NMR spectroscopy. Relaxation data were obtained at a spectrometer frequency of 600 MHz for 69 residues with an even spread along the parvalbumin polypeptide chain. A double approach that included Lipari‐Szabo analysis and direct mapping of spectral densities was used to interpret relaxation data in terms of internal dynamics. The former analysis provides valuable information about the overall rotational correlation time and S2 order parameters, while the mapping approach characterises the relative contributions of different motional frequencies. The results suggest that Ca2+‐loaded pike parvalbumin has a rigid structure, even in the functional regions, i.e., the Ca2+‐binding loops. The patterns of density‐function values are more sensitive to the secondary structure than those of S2. Moreover, depending on the sampling frequency, these patterns reveal different aspects of structure‐specific motions.
The Synthesis of mRNA in Isolated Mitochondria can be Maintained for Several Hours and is Inhibited by High Levels of ATP
Authors: Enríquez José A. , Fernández‐Silva Patricio , Pérez‐Martos Acisclo , López‐Pérez Manuel J. , Montoya Julio
Published: August 2004

The dependence for the maintenance of the synthesis and maturation of mitochondrial RNA on the supply of nucleo‐cytoplasmic factors has been investigated by a novel in organello RNA synthesis system. We found that mitochondrial DNA transcription can be maintained for several hours in isolated mitochondria. Analysis of the individual mitochondrial RNA species revealed that: the processing of the rRNA precursors and the stability of the mature rRNAs, but not the transcription itself, is severely impaired after short periods of incubation, indicating that these processes are strongly dependent on the mitochondrial interaction with the nucleo‐cytoplasmic compartment; the events that lead to the synthesis, processing and turnover of the mitochondrial mRNAs do not require the continuous supply of nucleo‐cytoplasmic factors, that are accumulated in excess by mitochondria. Furthermore, we present evidence indicating an inhibition of high ATP levels on the mitochondrial RNA polymerase activity, both in organello and in vitro. Consequently, it is proposed that mitochondrial mRNA synthesis can be regulated in response to changes in intramitochondrial ATP levels. This regulation of mitochondrial mRNA synthesis together with their very rapid turnover described here and elsewhere [Gelfand, R. & Attardi, G. (1981) Mol. Cell Biol. 1, 497–511], could represent a mechanism that would allow each individual mitochondrion to adjust its optimal levels of mRNA, and hence its translation capacity, in response to local energetic demands.
Calcium‐activated Opsin Phosphatase Activity in Retinal Rod Outer Segments
Authors: Kutuzov Mikhail A. , Bennett Nelly
Published: August 2004

We describe the presence in bovine retinal rod outer segments of a phosphatase which dephosphorylates phosphoopsin with an efficiency similar to that of PP2A, and which is stimulated by submicromolar levels of Ca2+ (half‐maximal activation, 0.4–0.5 μM). This enzyme is designated Ca2+‐activated opsin phosphatase (CAOP). CAOP has a molecular mass of 70–75 kDa as determined by gel filtration on Superose 12 and exhibits reversible Ca2+‐dependent oligomerization. An unidentified protein of approximately 25 kDa is necessary for full activity of CAOP and for cooperative binding of Ca2+ (h > 2). CAOP does not require Mg2+ and is inhibited by okadaic acid (median inhibitory concentration > 25 μM), which suggests that it is related to the PP1/2A/2B class of protein phosphatases. Like PP2B, CAOP is inhibited by trifluoperazine (median inhibitory concentration 40 μM), but calmodulin has no effect on CAOP activity, and CAOP is inhibited by mastoparan at much higher concentrations than PP2B. This combination of properties suggests that CAOP is not identical to any characterized protein phosphatase. Since the cytoplasmic concentration of Ca2+ in, retinal rod outer segments is reduced upon excitation by light, the existence of a Ca2+‐sensitive opsin phosphatase activity suggests that light‐dependent Ca2+ levels may control rhodopsin dephosphorylation.
Dephosphorylation of the Mannose‐6 Phosphate Recognition Marker is Localized in Late Compartments of the Endocytic Route
Authors: Bresciani Roberto , Von Figura Kurt
Published: August 2004

The mannose 6‐phosphate (Man6P) recognition marker in lysosomal proteins is known to be dephos‐phorylated after the delivery of lysosomal proteins to the endosome/lysosome compartment. The rate of Man6P recognition marker inactivation depends on the cell type and lysosomal protein. In the present study we show that in BHK 21 cells, which rapidly dephosphorylate lysosomal proteins, the recognition marker is stable in the endosomal compartment, to which lysosomal enzymes such as arylsulfatase A are delivered during endocytosis at 20°C. Dephosphorylation depends on the transfer of internalized lysosomal enzymes from the 20°C compartment to later compartments, most likely lysosomes. This transfer is sensitive to NH4Cl and nocodazole. In vitro experiments identified purple acid phosphatase (uteroferrin) as a candidate for the lysosomal phosphatase catalyzing in vivo the dephosphorylation of Man6P recognition marker.
Selection in vitro of Trans ‐Acting Genomic Human Hepatitis Delta Virus (HDV) Ribozymes
Authors: Nishikawa Fumiko , Kawakami Junji , Chiba Atsushi , Shirai Makoto , Kumar Penmetcha K. R. , Nishikawa Satoshi
Published: August 2004

In an effort to identify the functional structure as well as new active variants of the trans‐ acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. A total of 14 rounds of selection and amplification was repeated and various mutant ribozymes in G10 and G14 pools analyzed. Active ribozymes which were isolated in the present study (from G10 and G14) all possessed conserved bases (that were identified earlier) in the m‐acting molecule. A dominant clone G10‐68 variant was accumulated in generation 14. Interestingly, when base substitutions were analyzed in G10‐68 variant, we found that this variant appears to be close to antigenome‐like HDV ribozyme molecule. Further investigations of G10‐68 confirmed that each mutated base was the most appropriate nucleotide at every position of the HDV ribozyme.
Heterologous Expression and Site‐Directed Mutagenesis Studies on the Activation Mechanism and the Roles of the Basic Residues in the Prosegment of Aspergillopepsinogen I
Authors: Inoue Hideshi , Hayashi Takaharu , Huang Xiang‐Ping , Lu Jing‐Fang , Athauda Senarath B. P. , Kong Kwang‐Hoon , Yamagata Hideo , Udaka Shigezo , Takahashi Kenji
Published: August 2004

To study the structure/function relationship of the prosegment of aspartic proteinase, a putative pro‐form of aspergillopepsin I (or proteinase B) from Aspergillus niger var. macrosporus was expressed by Escherichia coli, refolded in vitro, and purified. The conversion of the purified proenzyme (aspergillopepsinogen I, proproteinase B) into the active mature form occurred at pH≤4.5 and was completely inhibited by pepstatin A, a specific inhibitor for aspartic proteinase, suggesting autoprocessing. The N‐terminus of this mature form was Glu67 (numbering in preproform), which was different from the N‐terminal Ser70 of native proteinase B although there was no significant difference in enzymatic activity. During the conversion, two intermediates were observed on SDS/PAGE, indicating a stepwise mechanism. The Lys56‐Phe57 sequence seems to be a counterpart of the Lys‐Tyr pair highly conserved in the prosequences of aspartic proteinases. When the mutant proenzyme (K56N), in which Lys56 was replaced with Asn by site‐directed mutagenesis, was allowed to refold under various conditions, no significant potential activity could be obtained.

Proproteinase B was also expressed by Bacillus brevis HPD31. This system required no in vitro refolding to obtain potentially active proenzyme, which was secreted into the culture medium (30–120mg/l) and had the same properties with that obtained by the E. coli system. The K56N mutant prepared by this system also had no potential activity, and was rapidly digested by incubation with native proteinase B, suggesting that the mutant did not fold correctly. On the other hand, the K56R mutant (Lys56→Arg) was potentially active. These results indicated that Lys56 is essential for the folding through electrostatic interaction with the catalytic Asp residues in the active site although it may be replaced with Arg. In the presence of a low concentration of pepstatin A, an incompletely processed form with N‐terminal Ser53 was obtained. Further, the R52Q (Arg52→Gln) mutant showed no processing but was converted to the active mature form by incubation with the native enzyme. Therefore, the cleavage between Arg52 and Ser53 is considered to be the initial and essential step of the autoactivation. The R26Q, K27Q, R36Q, K40Q, R42Q, and K66Q mutants were also potentially active. The K66Q mutant was processed to a form with N‐terminal Ala55.
An NMR‐Derived Model for the Solution Structure of Oxidized Thermotoga maritima 1[Fe4‐S4] Ferredoxin
Authors: Sticht Heinrich , Wildegger Gudrun , Bentrop Detlef , Darimont Beatrice , Sterner Reinhard , Rösch Paul
Published: August 2004

The solution structure of the 60‐residue 1[Fe4‐S4] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron‐sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1[Fe4‐S4] ferredoxins from Desulfovibrio gigas and Desulfovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the T. maritima protein. In particular, the T. maritima protein displayed a β‐sheet structure made up of strands located at the very NH2 and COOH termini of the protein, and an internal α‐helix. The internal α‐sheet observed in the D. gigas and D. africanus ferredoxins could not be confirmed in T. maritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure.
Different Exon–Intron Organizations of the Genes for Two Astacin‐Like Proteases, High Choriolytic Enzyme (Choriolysin H) and Low Choriolytic Enzyme (Choriolysin L), the Constituents of the Fish Hatching Enzyme
Authors: Yasumasu Shigeki , Shimada Hiraku , Inohaya Keiji , Yamazaki Ken , Iuchi Ichiro , Yasumasu Ikao , Yamagami Kenjiro
Published: August 2004

The hatching enzyme of the teleost, Oryzias latipes, is composed of two proteases, high choriolytic enzyme (choriolysin H, HCE) and low choriolytic enzyme (choriolysin L, LCE), which are similar in some enzymological characteristics and protein structure (55% identity in amino acid sequence) and belong to the astacin family. Two isoforms of HCE are detected. In the present study, the genes for HCE and LCE were isolated from the genomic library constructed from DNA of the inbred drR strain fish. In contrast to the close similarity of the enzymes, there was a marked difference in their gene organization. The LCE gene was a single copy gene and composed of eight exons interrupted by seven introns. The HCE genes were multicopy genes and lacked introns. In the haploid genome of the drR strain fish, there are eight HCE genes, seven of which were cloned. Each HCE gene was identified as that for either of the two isoforms of HCE. 5′ flanking regions of the LCE gene and the HCE genes had consensus TATA box sequences, but not CAT box nor GC box sequences. The big difference in the exon‐intron organization between the HCE genes and the LCE gene is discussed from an evolutionary viewpoint.
Photoaffinity Labeling of the Human Brain Cholecystokinin Receptor Overexpressed in Insect Cells
Authors: Gimpl Gerald , Anders Jonas , Thiele Christoph , Fahrenholz Falk
Published: August 2004

The human cholecystokinin B (CCKB) receptor was expressed in Sf 9 cells by infection with recombinant baculovirus. For immunodetection a c‐myc epitope tag (EQKLISEEDL) was fused at the aminoterminus of the CCKB receptor. In a second construct an additional hexa‐histidine tag was introduced at the C‐terminus of the CCKB receptor to enable employment of metal affinity chromatography. The two receptor constructs were expressed at densities of 6.0 ± 1.1 pmol/mg protein and 7.2 ± 1.1 pmol/mg protein, respectively which are 100–200‐fold higher compared with the receptor amounts found in natural sources. Saturation of the binding sites with [3H]propionyl‐CCK8 revealed Kd values of 4.5 ± 0.5 nM and 7.8 ± 0.6nM for the CCKB receptor without or with histidine tag. In SDS/PAGE and subsequent immunodetection the histidine‐tagged CCKB receptor migrated as a 55‐kDa band, whereas the CCKB receptor without C‐terminal modification revealed apparent molecular masses of 45 kDa and 49 kDa. The differences in the mass values observed for the two constructs suggest that the histidine tag could protect the CCKB receptor against proteolytical degradation from its C‐terminus. Furthermore two new photoreactive derivatives of cholecystokinin octapeptide residues 26–33 (CCK8) with high labeling efficiency and specificity for the cholecystokinin receptor subtype B were developed: [3H]BzBz‐des‐Met28‐[p ‐NH2Bz29]‐CCK8 and [3]BzBz‐biotinyl‐des‐Met28‐[p ‐NH2Bz29]‐CCK8. Both contain the p ‐benzoyl‐benzoyl (BzBz) residue at the N‐terminus for photoactivation and a p ‐aminobenzoyl (p ‐NH2BZ) residue instead of Met28‐Gly29 in cholecystokinin. Enzymatic deglycosylation of the CCKB receptor with N‐glycosidase F after photoaffinity labeling demonstrated that the CCKB receptor with three potential glycosylation sites was slightly glycosylated, amounting to a molecular mass of about 4 kDa. Using the biotinylated cholecystokinin derivative the photoaffinity‐labeled CCKB receptor could be purified 1260‐fold by a two‐step procedure including affinity chromatography on a streptavidin/avidin agarose matrix. For purification of the native receptor, an improved solubilization protocol for the CCKB receptor using dodecyl β‐D‐maltopyranoside was developed. The solubilized CCKB receptors with C‐terminal histidine tag retained their ligand binding characteristics after chromatography on a nickel affinity matrix.
Identification, cDNA Sequence and Deduced Amino Acid Sequence of the Mitochondrial Rieske Iron‐Sulfur Protein from the Green Alga Chlamydomonas reinhardtii
Authors: Atteia Ariane , Franzén Lars‐Gunnar
Published: August 2004

Specific oligonucleotide probes were used to isolate a cDNA clone for the mitochondrial Rieske iron‐sulfur protein of the green alga Chlamydomonas reinhardtii. The protein is synthesized as a longer precursor with a cleavable N‐terminal presequence of 54 amino acids but without a C‐terminal extension. Comparison of the predicted secondary structure of this N‐terminal sequence with that of the targeting signal of the chloroplast Rieske protein from C. reinhardtii [de Vitry (1994) J. Biol. Chem. 269, 7603–7609] indicates that, although they both have the potential to form amphiphilic α helices, the mitochondrial presequence may form a more hydrophobic helix that could penetrate deeper into the membrane. The N‐terminal part of the mature mitochondrial Rieske protein is characterized by a long, strongly hydrophilic N‐terminal domain and by a positive charge in the middle of the hydrophobic stretch that is presumed to interact with the bc1 complex. Thus, the protein from C. reinhardtii differs from the Rieske proteins from mammals or fungi.
Regulation of Prothymosin α During the Cell Cycle
Authors: Vareli Katerina , Tsolas Orestes , Frangou‐Lazaridis Maria
Published: August 2004

A number of studies have indicated that the small nuclear acidic protein prothymosin α is associated with cellular‐proliferation events. For example, c‐myc causes immediate transcriptional activation of prothymosin α, and prothymosin α antisense oligonucleotides inhibit myeloma cell division.

To investigate the regulation of prothymosin α, we examined its mRNA and protein levels during the cell cycle of mononuclear cells and fibroblastic cells. We isolated immunoreactive material from cellular extracts and immunolocalized the protein to the nucleus during the cell cycle. We report here that the material present in the cells is prothymosin α rather than the amino‐terminal peptide thymosin α1. [3H]Thymidine‐incorporation studies associate maximum accumulation of mRNA and protein with the S/G2 phase of the cell cycle. This induction of prothymosin α mRNA seems to resemble cyclin B expression and is more pronounced in fibroblasts. Moreover, transient‐transfection experiments indicate that transcription factor E2F is a strong positive regulator of the prothymosin α gene. Our results are consistent with the hypothesis that prothymosin α is involved in proliferation checkpoints of the cell cycle.
A Polyferredoxin with Eight [4Fe–4S] Clusters as a Subunit of Molybdenum Formylmethanofuran Dehydrogenase from Methanosarcina barkeri
Authors: Vorholt Julia A. , Vaupel Martin , Thauer Rudolf K.
Published: August 2004

Formylmethanofuran dehydrogenase (Fmd) from Methanosarcina barkeri is a molybdenum iron‐sulfur protein involved in methanogenesis. The enzyme contains approximately 30 mol non‐heme iron/mol and 30 mol acid‐labile sulfur/mol. We report here the cloning and sequencing of the encoding genes, and that these genes form a transcription unit fmdEFACDB. Evidence is provided that the subunit FmdB harbours the molybdenum‐containing active site and may bind one [4Fe–4S] cluster. fmdF encodes a protein with four tandemly repeated bacterial‐ferredoxin‐like domains and is predicted to be a polyferredoxin that could contain as many as 32 iron atoms in eight [4Fe–4S] clusters. The other genes code for proteins without sequence motifs characteristic for iron‐sulfur proteins. These findings suggest that most of the iron‐sulfur clusters present in the purified formylmethanofuran dehydrogenase are associated with the subunit FmdF. The finding that FmdF forms a tight complex with the other subunits of formylmethanofuran dehydrogenase indicates a function of the polyferredoxin in the reaction catalyzed by the enzyme. fmdE encodes a protein not present in the purified enzyme. All six genes of the fmd operon were expressed in Escherichia coli and yielded proteins of expected molecular masses. A malE‐fmdF gene fusion was constructed and expressed in E. coli, making the apoprotein of the polyferredoxin available in preparative amounts.
Secretory Non‐Pancreatic Phospholipase A2 and cyclooxygenase‐2 Expression by Tracheobronchial Smooth Muscle Cells
Authors: Vadas Peter , Stefanski Eva , Wloch Marek , Grouix Brigitte , Van Den Bosch Henk , Kennedy Brian
Published: August 2004

Lipid mediators of inflammation, contribute to airway hyper‐reactivity in asthma. Since production of lipid mediators is largely regulated by phospholipase A2 (PLA2), and since PLA2 expression in mesenchymal cells is induced by cytokines and other signals, we examined PLA2 expression by rat tracheobronchial smooth muscle cells (TBSMC). PLA2 expression in TBSMC cultures was markedly increased by tumour‐necrosis factor (TNF)α (130‐fold) and interleukin‐1β (IL‐1β) (7.4‐fold). Lipopolysaccharide (LPS; 100 ng/ml) resulted in a 51‐fold increase in extracellular PLA2 activity. PLA, expression by LPS‐stimulated or cytokine‐stimulated cells was downregulated by dexamethasone. Whereas forskolin or dibutyrl CAMP increased PLA2 activity, inhibition of protein kinase A but not tyrosine kinase reduced PLA2 expression. Northern blot analysis showed that TNFα and IL‐1β increased both PLA2 and inducible cyclooxygenase (Cox‐2) mRNA transcription. Addition of dexamethasone substantially blunted the increase in PLA2 and Cox‐2 mRNA. In contrast, the level of Cox‐1 mRNA was very low and did not change with the various treatments. Since proinflammatory lipid mediators have been implicated in the pathogenesis of asthma and PLA2 activity regulates generation of these lipid mediators, cytokine‐stimulated synthesis and release of PLA2 by airway smooth cells may contribute to the potentiation of airway inflammation in asthma.
Cloning, Sequencing and Expression in Escherichia coli of Two Rhizobium sp. Genes Encoding Haloalkanoate Dehalogenases of Opposite Stereospecificity
Authors: Cairns Stephen S. , Cornish Alex , Cooper Ronald A.
Published: August 2004

A 6.5‐kb EcoRI fragment of genomic DNA from a Rhizobium sp. cloned into pUC19 was able to endow Escherichia coli K‐12 with the novel ability to grow at the expense of 2‐chloropropionic acid. Subcloning showed that this property was a consequence of two dehalogenases encoded on a 2.2‐kb PstI fragment. Further subcloning of the PstI fragment led to two constructs that encoded, separately, dehalogenase activity that acted stereospecifically on D‐2‐chloropropionic acid and L‐2‐chlompropionic acid, respectively. The genes encoding these two stereospecific dehalogenases have been sequenced and shown to be separated by 177 bp of non‐coding DNA. Expression of the dehalogenase genes involved the vector promoter, suggesting that the anticipated Rhizobium sp. regulatory sequences were not functional in E. coli. Comparison of the deduced amino acid sequences of the two dehalogenases (18% identity) indicated that there was no obvious evolutionary relationship between (hem. Nor was there any striking identity with any other 2‐chlompropionic acid dehalogenase studied so far.
ATP Synthesis by the F0F1 ATP Synthase from Thermophilic Bacillus PS3 Reconstituted into Liposomes with Bacteriorhodopsin
Authors: Pitard Bruno , Richard Peter , Duñach Mireia , Rigaud Jean‐Louis
Published: August 2004

The correlation between the rate of ATP synthesis and light‐induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from therinophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light‐induced pH gradient. Maximal rates of ATP synthesis (up to 200 nmol ATP · min−1· mg protein–were obtained at saturating light intensities under a steady‐state pH gradient of about pH 1.25. It was detnonstrated that this was the maxmal pH attainable at 40°C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40°C. contributing to an increase in the rate of ATP synthesis, The H+/ATP stoichiometry was measured at the static‐head (equilbrium) conditions from the ratio of the phosphate potential to the size of the light‐induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient.

Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the inagnitude of pH remained the same or at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and irnpaired bacteriorhodopsin (Asp96→Asn mutaled bactericirliodopsin) further demonnstreted that this increase in the coupling efficiency could not he related to protein‐protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively chargcd phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transtnenibrane pH grudient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by dpH/Ψ transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light‐driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.
Haemolytic Rhodococcus equi Isolated from a Swine Lymph Node with Granulomatous Lesions
Authors: Pate M. , Pirš T. , Zdovc I. , Krt B. , Ocepek M.
Published: August 2004

Rhodococcus equi is generally thought to be non‐haemolytic although some earlier investigations reported minor haemolytic activity. A case of a haemolytic R. equi isolate from a swine lymph node with granulomatous lesions is described. This is a new contribution to knowledge of the cultural properties of R. equi.
Antimicrobial Resistance in Campylobacter spp., Escherichia coli and Enterococci Associated with Pigs in Australia
Authors: Hart W. S. , Heuzenroeder M. W. , Barton M. D.
Published: August 2004

The major influences on the amplification and spread of antibiotic‐resistant bacteria are the therapeutic use of antibiotics in human medicine and their use in livestock for therapy, prophylaxis and growth promotion. The use of veterinary antibiotics has many benefits to the livestock industries ensuring animal health and welfare, but use at subtherapeutic levels also exerts great selective pressure on emergence of resistant bacteria. The possible effect on human health is a problem of current debate. This study involved sampling pig carcasses, pig meat and assessing the level of resistance in zoonotic enteric bacteria of concern to human health. In South Australian pigs, thermophilic Campylobacter species showed widespread resistance (60–100%) to tylosin, erythromycin, lincomycin, ampicillin and tetracycline. No resistance was seen to ciprofloxacin. The enterococci demonstrated little resistance (0–30%) to vancomycin or virginiamycin, but the overall results from the antibiotic sensitivity testing of the enterococci have demonstrated how widespread their resistance has become. Escherichia coli strains showed widespread resistance to tetracycline and moderately common resistance (30–60%) to ampicillin and sulphadiazine. Resistance to more than one antibiotic was common. Pigs from New South Wales were also sampled and differences in resistance patterns were noted, perhaps reflecting different antibiotic use regimens in that state.
Female epispadias: are we missing the diagnosis?
Authors: Allen Lee , Rodjani Arry , Kelly Justin , Inoue Maki , Hutson John M.
Published: August 2004

OBJECTIVE

To describe three of four female patients with occult epispadias, as usually epispadias is reported to be a rare condition in females (1/480 000), and obvious and easy to diagnose, but less severe variants may still involve urethral sphincter incompetence in the absence of obvious clinical signs.

PATIENTS AND METHODS

The hospital records over a 10‐year period (1 January 1991 to 31 December 2000) were reviewed to find all female patients diagnosed with epispadias. Data were collected from the Consultative Council on Obstetric and Paediatric Mortality and Morbidity for the number of live births per year for the decade studied, and the incidence of female epispadias determined.

RESULTS

Over the 10‐year period there were 13 patients with a diagnosis of epispadias, from 636 698 total live births; four were female and nine male. Each of the female patients had varying degrees of epispadias; three had subtle signs with apparently normal external genitalia, and only one had a bifid clitoris. However, even the most minor variant case had a palpable notch in the symphysis pubis, and an ‘oblong’ external urethral meatus. Thus the incidence of female epispadias is at least 1/160 000.

CONCLUSION

Epispadias in females may be up to three times more common than previously expected, but even experienced clinicians can miss the diagnosis. The importance of a careful examination should be emphasized, especially in female patients with chronic wetting of unknown cause. A bifid clitoris is an important sign but not always present. Key diagnostic features include drug‐resistant wetting, a very low leak pressure on cystometrography, a palpable gap in the pubic symphysis and an abnormal oval‐shaped external urethral meatus.
Urinary continence after salvage radiation therapy following radical prostatectomy, assessed by a self‐administered questionnaire: a prospective study
Authors: Fontaine Eric , Ben Mouelli Sami , Thomas Laurent , Otmezguine Yves , Beurton Daniel
Published: August 2004

OBJECTIVE

To evaluate urinary continence after salvage radiotherapy following radical prostatectomy (RP) for clinically localized prostate cancer.

PATIENTS AND METHODS

In all, 145 men had a retropubic RP in our department between 1992 and 2000. From this group, salvage radiotherapy with the dose of 65 Gy was given to 18 patients for a rising prostatic specific antigen (PSA) level. The mean (range) age at RP was 63 (50–72) years and the initial PSA level 14.95  (4.8–34) ng/mL. The radiotherapy was given at 46 (6–96) months after RP. A self‐administered questionnaire about their urinary status was mailed to the patients before and 18 months after radiotherapy. The mean follow‐up after radiotherapy was 34 (20–70) months.

RESULTS

Before radiotherapy, 17 patients were continent (defining continence as no regular use of pads). After salvage radiotherapy, 16 men had had no change in their urinary status, even for the one patient using pads. One patient with stress urinary incontinence showed a slight worsening of his urinary status after radiotherapy. Another patient who was continent before radiotherapy developed urgency with no urinary leakage. Fourteen men stated that they were very satisfied or satisfied about their urinary status after radiotherapy and four were mildly satisfied. Nine would undergo radiotherapy again even with their present continence status and nine probably would.

CONCLUSION

Using an anonymous self‐administered questionnaire, salvage radiotherapy for a rising PSA level seems to be safe and does not worsen the continence achieved after RP in most patients.
Predictors of prostate cancer on extended biopsy in patients with high‐grade prostatic intraepithelial neoplasia: a multivariate analysis model
Authors: Abdel‐Khalek Mohamed , El‐Baz Mahmoud , Ibrahiem El‐Houssieny
Published: August 2004

OBJECTIVE

To define the importance of extended biopsy in patients with high‐grade prostatic intraepithelial neoplasia (HGPIN) and to define predictors of cancer in extended biopsy in patients with HGPIN, using multivariate analysis.

PATIENTS AND METHODS

In all, 83 patients with previous sextant biopsy of HGPIN had an extended 11‐core biopsy taken. Patients with a negative biopsy for cancer were followed by serum prostate‐specific antigen (PSA) and digital rectal examination (DRE) every 6 months. The extended biopsy was repeated in 21 patients. The criteria for second biopsy were an increase in PSA and/or abnormal changes on DRE. Overall, 49 patients had a transurethral resection of the prostate (TURP). The cancer‐detection rate on extended biopsy was correlated with risk factors using the chi‐square test and multivariate analysis.

RESULTS

Extended biopsy detected prostate cancer in 30 of the 83 men (36%), with positive cores in only 20 sextant biopsy sites (67%), in only seven in additional sites (23%), and both in three (10%). Of the 21 patients who had repeat extended biopsy, four (19%) had cancers. There were two carcinomas in the 49 TURP specimens (4%). The PSA level, DRE and transrectal ultrasonography findings were not predictive of cancer in extended biopsies (chi‐square test). Patient age, PSA density and the number of cores with HGPIN (all P < 0.001) had a significant effect on the cancer‐detection rate, and multivariate analysis showed that all three were independent predictors of cancer. A logistic regression model was designed to predict the probability of cancer in extended biopsy, with an overall accuracy of 78%.

CONCLUSION

Extended biopsy improved the cancer detection rate by 23% in patients with HGPIN. Patient age, PSA density and the number of cores with HGPIN were the only independent predictors of cancer.
Prostate size influences the outcome after presenting with acute urinary retention
Authors: McNeill Alan S. , Rizvi Syed , Byrne Derek J.
Published: August 2004

The relevance of prostatic size to treatment outcome has been stressed by many authors, in terms of likelihood of response to 5α‐reductase inhibitors and as a risk factor for future surgery or acute urinary retention. Authors from Scotland studied patients who developed acute retention and were treated with catheter removal and α‐adrenergic blockage. They found that, in this group of patients, prostate size was the key determinant as to whether surgery would be required or whether the more conservative treatment would be successful.

OBJECTIVE

To evaluate the long‐term outcome in an open follow‐up of a cohort of patients who had had a successful trial without catheter (TWOC) after an episode of acute urinary retention (AUR), as it is now widely accepted that giving an α‐blocker, e.g. alfuzosin, increases the success rate of TWOC.

PATIENTS AND METHODS

In this prospective trial, 81 patients with a first episode of AUR related to benign prostatic obstruction received either sustained‐release alfuzosin (40) 5 mg twice daily or placebo (41) for 48 h. The catheter was removed after 24 h of treatment and the patient's ability to void assessed. Those who voided successfully entered an open follow‐up, the defined endpoints of which were the date of recurrent AUR, date of bladder outlet surgery, date of last follow‐up or death, and factors that influenced the long‐term outcome after a successful TWOC were examined.

RESULTS

Of the 34 patients who had a successful TWOC (22 on alfuzosin, 12 placebo, P= 0.03), 21 continued on an α‐blocker at the discretion of their urologist. In all, 26 had a further episode of AUR or surgery during the 6‐year follow‐up. The mean (median, range) time to the second episode of AUR in the 20 (59%) patients affected was 1.4 (0.6, 0–5.95) years. Nineteen (56%) men had bladder outlet surgery, 13 after a second episode of AUR. The mean time to operation after the first AUR was 1.85 (1.1, 0.04–5.4) years. The remaining eight (24%) patients remained free of further AUR and surgery. The size of the prostate assessed on a digital rectal examination by the admitting urologist was the only factor with a significant effect on the long‐term outcome. A postvoid residual of > 50 mL was associated with a greater likelihood of recurrent AUR or surgery, but this was not statistically significant.

CONCLUSIONS

This study provides further evidence of the importance of prostate size as a prognostic factor in determining the outcome in patients with prostatic obstruction. Whilst most men presenting with AUR will eventually have prostatic surgery, a significant minority will not. An assessment of risk factors such as prostate size may identify those who require urgent intervention after a successful TWOC. The role of continued medical therapy with α‐blockers and/or 5α‐reductase inhibitors after a successful TWOC merits further investigation.
Antidiuretic hormone in elderly male patients with severe nocturia: a circadian study
Authors: Moon Du Geon , Jin Myeong Heon , Lee Jeong Gu , Kim Je Jong , Kim Myung Gon , Cha Dae Ryong
Published: August 2004

OBJECTIVE

To investigate the circadian variation of plasma antidiuretic hormone (ADH) and urine output in patients with severe nocturia (> three times per night) and to assess the effect of oral desmopressin on nocturnal urine output in these patients.

PATIENTS AND METHODS

Twelve patients with severe nocturia and five age‐matched controls without were assessed over 24 h (circadian sampling) during a 72‐h hospital admission. Blood levels of ADH and changes of urine output were measured in the patients before and after the oral administration of desmopressin (0.2 mg, at 22.00 hours in the second day), and in the controls not treated with desmopressin.

RESULTS

Compared with the normal control, the patients had no diurnal variation in urine output and greater nocturnal urine production, associated with a lack of nocturnal increase in ADH level. Compared with the baseline urine output, desmopressin significantly decreased night‐time (23.00–08.00 hour) urine output in the patients (P < 0.05). Desmopressin significantly increased the osmolality of night‐time urine (P < 0.05), and there was no systemic adverse reaction.

CONCLUSIONS

Severe nocturia in a large proportion of elderly men with lower urinary tract symptoms is caused by nocturnal polyuria and natriuresis, because they have no nocturnal increase in ADH. These results suggest that desmopressin may be effective in decreasing nocturnal urine production in patients with severe nocturia who do not respond to conventional treatment.