bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular Processing Limits the Heterologous Expression of Secretory Component in Mammalian Cells
Authors: Cottet Sandra , Corthésy Blaise
Published: July 2004

Recombinant vaccinia‐virus‐based expression systems are very popular for the overproduction of proteins in mammalian cell lines. Both the double virus T7/vaccinia hybrid system and the single recombinant strategy based on the p11 K late promoter were evaluated for their ability to govern expression and secretion of recombinant human secretory component (SC), a glycoprotein associated with IgA in mucosal secretions. We report here that, while the T7 promoter is transcriptionally 3.4‐fold more active than the p11 K promoter, no difference in levels of secreted recombinant human SC is observed using either vaccinia system to infect CV‐1 cells. High transcription, and thus translation levels, lead to saturation of early processing steps involved in protein export. Both systems exhibit transient accumulation of comparable amount of recombinant human SC in the endoplasmic reticulum and/or the cis Golgi network, as demonstrated by immunofluorescence and endoglycosidase H (EndoH) sensitivities. Exposure of infected cells to tunicamycin results in similar inhibition of recombinant human SC export, further arguing that N‐linked glycosylation is necessary for proper folding and subsequent secretion. Moreover, pulse‐chase experiments indicate that newly synthesized recombinant human SC is not completely processed in a mature glycoprotein and that a portion of overexpressed SC might be degraded before it can be secreted. Recombinant human SC behaves identically to native SC in terms of kinetics of secretion and IgA‐binding capacity.

Our results indicate that optimization of expression systems should not only rely on the design of effective vectors, but also on the identification and clearance of the cellular bottlenecks associated with maturation of the secreted proteins.
Carbohydrate Protection of Enzyme Structure and Function against Guanidinium Chloride Treatment Depends on the Nature of Carbohydrate and Enzyme
Authors: Sola‐Penna Mauro , Ferreira‐Pereira Antônio , Lemos Adriana dos Passos , Meyer‐Ferwandes José Roberto
Published: July 2004

Baker's yeast cells accumulate osmolytes as a response to several stress conditions such as hightemperature and low‐temperature shifts, dehydration, or osmotic stress. One of the major osmolytes that accumulates is trehalose, which plays an essential role affecting the survival of yeast at the time of stress. In this report, we show that trehalose efficiently protects the function and the structure of two yeast cytosolic enzymes against chemical denaturation by guanidinium chloride. Other sugars tested also protected yeast pyrophosphatase and glucose‐6‐phosphate dehydrogenase structure against guanidinium chloride effects, but were not as efficient at protecting enzyme activity. The thermostable pyrophosphatase from Bacillus stearothermophilus was also protected by several sugars against the chaotropic properties of guanidinium chloride, but was only protected by trehalose against functional inactivation. The function of the membrane‐embedded H+‐ATPase from yeast could not be protected by any of the tested sugars, although all of the sugars protected its structure from guanidinium‐chloride‐induced unfolding. The results presented in this study suggest that several sugars are able to prevent protein unfolding induced by a chaotropic compound. However, prevention of functional inactivation depends on the nature of the sugar. Trehalose was the most efficient, being able to protect many cytosolic enzymes against guanidinium chloride. The efficiency of protection also depended on the nature of the protein tested. This might explain why trehalose is one of the osmolytes accumulated in yeast and also why it is not the only osmolyte to accumulate.
Cloning of the Semenogelin II Gene of the Rhesus Monkey
Authors: Ulvsbäck Magnus , Lundwall Åke
Published: July 2004

The semenogelin II gene from the rhesus monkey has been cloned and characterized. The transcription unit is split into three exons of 97, 2086 and 124 bp, with two intervening introns of 241 bp and 862 bp. The first exon codes for a 23‐amino‐acid signal peptide and the two amino‐terminal residues of the secreted protein. The second exon codes for the rest of the mature protein, and the third exon contains non‐coding nucleotides only. Secreted rhesus monkey semenogelin II consists of 683 amino acid residues, has a calculated Mr of 77362, is devoid of Cys and Met, and displays a highly repetitive structure composed of ten 60‐amino‐acid repeats. Hybridization with genomic DNA showed that the semenogelin II gene of man, rhesus monkey and baboon has evolved through extension of the coding region with 360‐bp segments. In contrast, the length of the semenogelin I gene of these species appears to be conserved. The two genes are also present in some New World monkeys, as was revealed by hybridization with genomic DNA from the marmoset. However, another New World monkey, the cotton‐top tamarin, carries only one semenogelin gene, but also has a gene that is similar to the mouse semenoclotin gene.
Quantitative Analyses of Myosin Heavy‐Chain mRNA and Protein Isoforms in Single Fibers Reveal a Pronounced Fiber Heterogeneity in Normal Rabbit Muscles
Authors: Peuker Heidemarie , Pette Dirk
Published: July 2004

A highly sensitive method of reverse‐transcriptase polymerase chain reaction (RT‐PCR) was established to study myosin heavy‐chain (MHC) mRNA isoform expression in single fibers of rabbit limb muscles. In combination with myofibrillar adenosine triphosphatase histochemistry and electrophoretic separation of MHC protein isoforms in fragments of the same fibers, the direct RT‐PCR method identified the pMHC20‐40 and pMHC24‐79 cDNA sequences as being specific to MHCIIb and MHCIId/x isoforms, respectively. In addition, a direct RT‐PCR was established for determining relative amounts of MHC mRNA isoforms by using a sequence specific to α‐skeletal actin as an endogenous reference. Analyses of large amounts of single fibers revealed an unexpected heterogeneity of the fast fiber population with regard to numerous fibers coexpressing MHCIIb and MHCIId/x. Based on quantitative RT‐PCR, the percentages of MHCIIb/MHCIId hybrid fibers amounted to approximately 55% in the deep portion of gastrocnemius, to 43% in the adductor magnus, and to 12% in psoas muscle. Moreover, the two MHC mRNA isoforms were nonuniformly distributed along the fiber length. Qualitative RT‐PCR detected even higher amounts of hybrid fibers in the three muscles. The percentages of hybrid fibers identified at the protein level were smaller in adductor magnus muscle (25%) and psoas muscle (5%), but equaled that of the mRNA analysis in gastrocnemius muscle (61%). The detection of high amounts of IIBD and IIDB fibers suggested that hybrid fibers represent functional elements within the fiber spectrum of normal muscles. Our observations on hybrid fibers reveal a heterogeneity within the fiber population of normal muscles that has not been realized to date.
Subcellular and Tissue Distribution, Partial Purification, and Sequencing of Calmodulin‐Stimulated Ca2+‐Transporting ATPases from Barley (Hordeum Vulgare L.) and Tobacco (Nicotiana Tabacum)
Authors: Dainese Paola , James Peter , Baldan Barbara , Carafoli Ernesto
Published: July 2004

The subcellular distribution of plasma‐membrane‐type Ca2+‐transporting ATPases was studied in barley leaves (Hordeum vulgare L.). A highly enriched plasma membrane (PM) fraction was analysed for Ca2+ pumps and compared with several inner‐membrane preparations, including the tonoplast, the envelope of the chloroplast, and an endoplasmic reticulum (ER)‐enriched fraction. The enzymes were identified and characterised with regard to the phosphointermediate formation, their nucleotide specificity and their binding to calmodulin. A Ca2+‐transporting ATPase (molecular mass ≈ 130 kDa), which showed high specificity for ATP and high affinity for calmodulin, was localised in the PM. A 116‐kDa Ca2+‐transporting ATPase, probably located in the ER, showed lower nucleotide specificity and weaker affinity for calmodulin. A comparison of the distribution of the pumps in leaves and roots indicated that the ratio of expression of the two enzymes changed in a tissue‐specific manner: the PM pump was dominant in leaves, while the inner‐membrane pump was expressed at a higher level in the roots.

For the purification of calmodulin‐binding proteins (Ca2+ pumps), microsomes isolated from tobacco cell cultures were used. Two active Ca2+ pumps were identified, and the one at 116kDa was partially sequenced.
Room Temperature Phosphorescence Study of Phosphate Binding in Escherichia Coli Alkaline Phosphatase
Authors: Sun Li , Kantrowitz Evan R. , Galley William C.
Published: July 2004

The phosphorescence spectrum and decay of Trp109 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21°C. Changes in the spectrum and decay from the steady‐state in response to non‐covalent phosphate binding suggested a phosphate‐induced alteration in the local environment surrounding Trp109 which lies buried below the active site. The seemingly inflexible structure in the region of Trp109, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate. However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits. For ratios of phosphate/enzyme (mol/mol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half‐saturated and fully‐saturated enzymes consistent with observed enzyme turnover rates. The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 ± 0.1 μM. The non‐exponential decays at P/Ed (phosphate/dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate‐free and half‐saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half‐saturated molecule cannot be contributing significantly to catalysis under steady‐state conditions. The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half‐saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.
Selective Fast Degradation of Cytochrome P‐450 2E1 in Serum‐Deprived Hepatoma Cells by a Mechanism Sensitive to Inhibitors of Vesicular Transport
Authors: Zhukov Andrei , Ingelman‐Sundberg Magnus
Published: July 2004

Cytochrome P‐450 2E1 (CYP2E1) is characterized by a rapid turnover in the liver and some cell lines and the ability of substrates and heme iron ligands to inhibit significantly enzyme degradation. In the Fao hepatoma cell line, CYP2E1 was found to be fairly stable (half‐life of 26 h), but serum withdrawal resulted in its rapid disappearance from the microsomal fraction (half‐life of about 7 h) as evaluated using cycloheximide chase. The effect of serum withdrawal could be partially reversed by the addition of albumin to the culture medium, whereas insulin and the insulin‐like growth factor IGF‐I had no additional effect. The effect of serum withdrawal was specific for CYP2E1 since (a) no concomitant fast degradation of CYP2B1 and NADPH–cytochrome P‐450 reductase was observed and (b) the CYP2E1 ligands ethanol and imidazole prevented the fast degradation of the enzyme. The lysosomotropic agent ammonium chloride and the inhibitor of autophagocytosis 3‐methyladenine slowed down CYP2E1 degradation by about 30%, while leupeptin had no effect. Under the same conditions, the degradation of total long‐lived cell protein showed the same sensitivity to ammonium chloride, but was significantly less sensitive to 3‐methyladenine and serum and not sensitive to ethanol and imidazole. CYP2E1 degradation was inhibited by combined treatment with brefeldin A and nocodazole, which blocks both anterograde and retrograde vesicular transport between endoplasmic reticulum and the Golgi apparatus. The data point to the existence of a selective mechanism for the degradation of membrane proteins in serum‐deprived cells in addition to nonselective autophagocytosis. The selective degradation of CYP2E1 may be attained by means of its selective vesicular transport to an acidic post‐endoplasmic reticulum compartment.
The N‐Terminal Arg‐Rich Region of Human Immunodeficiency Virus Types 1 and 2 and Simian Immunodeficiency Virus Nef is Involved in RNA Binding
Authors: Echarri Asier , González Maria Eugenia , Carrasco Luis
Published: July 2004

Comparison of the amino acid sequences of human immunodeficiency virus (HIV) Nef protein and several RNA‐binding proteins shows similarities in some regions of these proteins. Thus, poliovirus protein 2C, an RNA‐binding protein, shares with Nef the sequence YXQQ…MDD…DXXD. In addition, both proteins contain an Arg‐rich motif that, in the case of poliovirus 2C, is involved in RNA‐binding activity. Moreover, the RNA‐binding, and‐terminator N proteins of λ, Φ21 and P22 phages show sequence similarities with HIV Nef at the Argrich motif. To assess the significance of this motif, native and deletion variants of Nef protein were assayed for RNA‐binding activity. The N‐terminal 35 amino acids of HIV‐1 Nef that comprise the Arg‐rich motif are sufficient for RNA binding. Point mutations engineered at the Arg‐rich motif of HIV‐1 Nef revealed that basic amino acid residues are essential for RNA‐binding activity. The Nef proteins from HIV‐2 and SIV can also interact with RNA, while the same proteins with the N‐terminal Arg‐rich domain truncated fail to interact with RNA. These findings indicate that all three Nef proteins from HIV‐1, HIV‐2 and simian immunodeficiency virus belong to the RNA‐binding family of proteins. The three proteins contain an Arg‐rich region at the N‐terminus which is necessary to interact with RNA.
Differential Interaction of Bile Acids from Patients with Inborn Errors of Bile Acid Synthesis with Hepatocellular Bile Acid Transporters
Authors: Stieger Bruno , Zhang Jie , O'Neill Brigitte , Sjövall Jan , Meier Peter J.
Published: July 2004

People with genetic or acquired defects in the biosynthesis of bile acids may suffer from cholestasis. Patients with a deficiency of 3β‐hydroxy‐Δ5‐C27‐steroid dehydrogenase/isomerase form 3β,7α‐dihydroxy‐and 3β,7α,12α‐trihydroxy‐5‐cholenoic acids, the sulfated and partially glycine‐conjugated forms of which are found in their urine and bile. 3‐Oxo‐Δ4 bile acids are detected in the urine of patients with a deficiency of 5β‐reductase. It has been postulated that these unusual bile acids might act as cholestatic agents in these patients.

The aim of the present study was to test this hypothesis in an in vitro system, since the abnormal bile acids would be metabolized in in vivo experiments. Basolateral (sinusoidal) and canalicular plasma membrane vesicles were isolated from rat liver. A rapid filtration method was used to determine transport of cholyltaurine in the presence of model bile acids into the isolated vesicles. It was found that 3β,7α‐dihydroxy‐5‐cholenoic acid and 7α‐hydroxy‐3‐oxo‐4‐cholenoic acid both inhibited the apical, ATP‐dependent transport system for cholyltaurine in a competitive manner with Km values of 15 μM and 16 μM, respectively. Radioactively labeled 3β,7α‐dihydroxy‐5‐cholenoyltaurine and 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine were not transported by the same transport system. The same types of experiments were performed with basolateral plasma membrane vesicles. It was found that, in contrast to the canalicular ATP‐dependent bile acid transport system, only 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine was a competitive inhibitor of the sodium‐dependent transport system for cholyltaurine with a Km of 16 μM. Studies with radioactively labeled 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine and 3β,7α‐dihydroxy‐5‐cholenoyltaurine revealed that 7α‐hydroxy‐3‐oxo‐4‐cholenoyltaurine was transported in a sodium‐dependent manner into basolateral rat liver plasma membrane vesicles, whereas 3β,7α‐dihydroxy‐5‐cholenoyltaurine was not transported in a sodium‐dependent way.

These results support the hypothesis that the unusual bile acids found in patients with defects in bile acid biosynthesis might act as cholestatic agents by inhibiting the canalicular ATP‐dependent transport system for bile acids which constitutes the rate‐limiting step in the overall process of bile acid transport across hepatocytes. Furthermore, the experiments demonstrated that, despite similar substrate specificities, the basolateral sodium‐dependent and the apical ATP‐dependent transport system for cholyltaurine might have different recognition sites for bile acids.
Uncoupling of Actomyosin Adenosinetriphosphatase by Heparin and Its Fragments
Authors: Tersariol Ivarne L. S. , Dietrich Carl P. , Nader Helena B.
Published: July 2004

Heparin and its enzymatic fragments, prepared by degradation of heparin with heparinase from Flavobacterium heparinum, were capable of inhibiting the actomyosin‐ATPase activity obtained from striated and smooth vascular muscles. Heparin did not inhibit the myosin‐ATPase activity in absence of actin. The results show that heparin changes the step of ATP hydrolysis of the complex actomyosin‐ATPase by uncoupling the conformational transition on the myosin‐head induced by actin upon the nucleotide‐binding site. This mechanism is cooperative and dependent on conformational states of actomyosin complex which in turn is regulated by ATP and calcium levels. It was observed that in the presence of ATP, actin does not compete with heparin for binding to myosin showing that heparin and actin have different binding sites on myosin. The binding of heparin and ATP is cooperative suggesting that the nucleotide binding leads to an exposition of a second heparin‐binding site. However, in the absence of ATP, actin competes with heparin for a binding site on the myosin. These results strongly suggest that in the weakly binding state of actin to myosin, the binding of heparin is powerful and in the rigor state its binding is decreased.
The Role of CCAAT/Enhancer‐Binding Protein α and a Protein that Binds to the Activator‐Protein‐1 Site in the Regulation of Liver‐Specific Expression of the Winter Flounder Antifreeze Protein Gene
Authors: Chan Shing‐Leng , Miao Ming , Fletcher Garth L. , Hew Choy L.
Published: July 2004

Winter flounder, Pleuronectes americanus, produces antifreeze proteins (AFPs) to avoid freezing during winter. The AFP in the blood is synthesized by the liver and its annual level is regulated by environmental factors and somatotropin. The only intron (+106 to +602) of the AFP gene contains putative binding sites for liver‐enriched transcription activators. Due to the lack of an appropriate fish liver cell line, a mammalian model system was used to demonstrate the liver‐specific enhancer activity of the intron. Deletion analysis of the intron indicated nucleotide sequences between +192 bp and +334 bp were important for the enhancer activity. Further refinement showed that the element B (+303 to +322 bp) resided in this region interacted with proteins from rat nuclear extracts by DNase I footprinting and mobility shift analysis. Gel retardation with competition and supershift experiments defined element B to include binding sites for CCAAT/enhancer binding protein α(C/EBPα), a liver‐enriched transcription factor, and a novel activator protein‐1 (AP‐1) site binding protein, designated as antifreeze enhancer binding protein (AEP). Residues important to DNA‐protein interaction in element B were mapped by methylation interference. Mutations in element B by site‐directed mutagenesis destroyed the enhancer activity of the intron. Ultraviolet crosslinking experiments using element B revealed the specific binding of two proteins of approximately 43 and 80 kDa. The present findings provide a mechanism(s) by which the tissue specificity of AFP gene expression can be achieved. Integration of the present information with the somatotropin‐mediated signal transduction pathways has generated a working model for the hormonal regulation and seasonal expression of the AFP gene.
Antioxidants As Well As Oxidants Activate C‐fos Via Ras‐Dependent Activation of Extracellular‐Signal‐Regulated Kinase 2 and Elk‐1
Authors: Müller Judith M. , Cahill Michael A. , Rupec Rudolf A. , Baeuerle Patrick A. , Nordheim Alfred
Published: July 2004

Intracellular reactive oxygen intermediate (ROI) levels play an important role in numerous physiological and pathophysiological conditions. Apart from causing oxidative stress and damage, ROI changes differentially activate gene expression. However the proto‐oncogene encoding the AP‐1 transcription factor subunit c‐Fos is induced by both prooxidants and antioxidants. Here, the transcription factor Elk‐1 is identified as being responsible for c‐fos serum response element (SRE) induction in response to changes in the cellular redox status induced by treatment with either the oxidant H2O2 or various structurally unrelated antioxidants. A temporal correlation is observed between changes in the phosphorylation status of Elk‐1 and the activation of the mitogen‐activated protein kinase 2 (ERK2) in response to cellular redox changes. Correspondingly, the transcriptional response of the SRE to redox fluctuations is attenuated upon mutation of critical ERK2 target residues within the Elk‐1 transactivation domain to alanine. Signals elicited by antagonistic intracellular redox changes converge at or above the level of Ras or an effector of Ras, leading to similar activation of c‐fos transcription, since an [N17]Ras mutant interfered with redox signaling. Hence components of signaling pathways are revealed to be shared by mitogenic and redox‐dependent stimuli.
Sequencing and Expression of the Gene Encoding a Cold‐Active Citrate Synthase from an Antarctic Bacterium, Strain DS2‐3R
Authors: Gerike Ursula , Danson Michael J. , Russell Nicholas J. , Hough David W.
Published: July 2004

The gene encoding citrate synthase from a novel bacterial isolate (DS2‐3R) from Antarctica has been cloned, sequenced and over expressed in Escherichia coli. Both the recombinant enzyme and the native enzyme, purified from DS2‐3R, are cold‐active, with a temperature optimum of 31°C. In addition the enzymes are rapidly inactivated at 45°C, and show significant activity at 10°C and below. Comparison of amino acid sequences indicates that DS2‐3R citrate synthase is most closely related to the enzyme from gram‐positive bacteria. The amino acid sequence of the DS2‐3R enzyme shows several features previously recognised in other cold‐active enzymes, including an extended surface loop, an increase in the occurrence of charged residues and a decrease in the number of proline residues in loops. Other changes observed in some psychrophilic enzymes, such as a decrease in isoleucine content and in arginine/(arginine+lysine) content, were not seen in this case.
Cloning of an Annelid Fibrillar‐Collagen Gene and Phylogenetic Analysis of Vertebrate and Invertebrate Collagens
Authors: Sicot François‐Xavier , Exposito Jean‐Yves , Masselot Monique , Garrone Robert , Deutsch Jean , Gaill Françoise
Published: July 2004

Arenicola marina possesses cuticular and interstitial collagens, which are mostly synthesised by its epidermis. A cDNA library was constructed from the body wall. This annelid cDNA library was screened with a sea‐urchin‐collagen cDNA probe, and several overlapping clones were isolated. Nucleotide sequencing of these clones revealed an open reading frame of 2052 nucleotides. The translation product exhibits a triple helical domain of 138 Gly‐Xaa‐Yaa repeats followed by a 269‐residue‐long C‐terminal non‐collagenous domain (C‐propeptide). The triple helical domain exhibits an imperfection that has been previously described in a peptide produced by cyanogen bromide digestion (CNBr peptide) of A. marina interstitial collagen. This imperfection occurs at the same place in the interstitial collagen of the vestimen‐tiferan Riftia pachyptila. This identifies the clone as coding for the C‐terminal part of a fibrillar collagen chain. It was called Fam1α, for fibrillar collagen 1α chain of A. marina. The non‐collagenous domain possesses a structure similar to carboxy‐terminal propeptides of fibrillar pro‐α chains. Only six conserved cysteine residues are observed in A. marina compared with seven or eight in all other known C‐propeptides. This provides information on the importance of disulfide bonds in C‐propeptide interactions and in the collagen‐assembly process. Phylogenetic studies indicate that the fibrillar collagen 1α chain of A. marina is homologous to the R. pachyptila interstitial collagen and that the Fam1α gene evolved independently from the other a‐chain genes. Complementary analyses indicate that the vertebrate fibrillar collagen family is composed of two monophyletic subgroups with a specific position of the collagen type‐V chains.
Purification and Characterization of a Mitochondrial, Single‐Stranded‐DNA–Binding Protein from Paracentrotus Lividus Eggs
Authors: Roberti Marina , Musicco Clara , Loguercio Polosa Paola , Gadaleta Maria Nicola , Quagliariello Ernesto , Cantatore Palmiro
Published: July 2004

A binding protein for single‐stranded DNA was purified from Paracentrotus lividus egg mitochondria to near homogeneity by chromatography on DEAE‐Sephacel and single‐stranded‐DNA–cellulose. The protein consists of a single polypeptide of about 15 kDa. Glycerol gradient sedimentation analysis suggested that P. lividus mitochondrial single‐stranded‐DNA–binding protein exists as a homo‐oligomer, possibly a tetramer, in solution. The protein shows a stronger preference for poly(dT) with respect to single‐stranded M13, poly(dI) and poly(dC). Binding to poly(dA) takes place with much lower affinity. The binding‐site size, determined by gel mobility‐shift experiments with oligonucleotides of different length, is approximately 45 nucleotides. The binding to single‐stranded DNA occurs with low or no cooperativity and is not influenced by ionic strength. The protein has a very high affinity for the DNA: its apparent macroscopic association constant is 2×109 M−1, a value which is the highest among the mitochondrial single‐stranded‐DNA–binding proteins characterized to date. The lack of cooperativity and the high association constant represent distinctive features of this protein and might be related to the peculiar mechanism of sea urchin mitochondrial DNA replication.
The Aminonucleoside Antibiotic A201A is Inactivated by a Phosphotransferase Activity from Streptomyces Capreolus NRRL 3817, the Producing Organism
Authors: Barrasa María Inmaculada , Tercero José Antonio , Jimenez Antonio
Published: July 2004

A novel resistance determinant (ard2) to the aminonucleoside antibiotic A201A was cloned from Streptomyces capreolus NRRL 3817, the producing organism, and expressed in Streptomyces lividans. Sequencing and subcloning experiments of a 3‐kb fragment localized ard2 to an ORF of 591 nucleotides. Cell‐free extracts from both S. capreolus and S. lividans (ard2) were shown to phosphorylate A201A in an ATP‐dependent reaction. The resulting product (P‐A201A) was purified and shown to lack any detectable biological activity against a gram‐positive indicator organism. Phosphorylation by Ard2 takes place on the hydroxyl group at C2 of the unsaturated hexofuranose moiety of A201A, as shown by 1H‐NMR analysis of purified P‐A201A. The expression of ard2 appears to be developmentally controlled. In addition to ard2, the sequenced DNA fragment contained two incomplete ORFs (2 and 5) and one complete ORF (4), which appear to determine enzymes of the A201A biosynthetic pathway. Whereas the deduced product of ORF2 did not show any similarity to proteins in data banks, those of ORF5 and ORF4 encode putative glycosyltransferase and ketoreductase activities, respectively. ard2 and these three ORFs seem to be transcribed in a single polycistronic transcript, which supports the notion that they are a part of an A201A biosynthetic gene cluster.
An Internal FK506‐Binding Domain is the Catalytic Core of the Prolyl Isomerase Activity Associated with the Bacillus Subtilis Trigger Factor
Authors: Göthel Sven F. , Schmid Roland , Wipat Anil , Carter Noel M. , Emmerson Peter T. , Harwood Colin R. , Marahiel Mohamed A.
Published: July 2004

Two major families of peptidylprolyl cis‐trans‐isomerases, the cyclophilins and the structurally unrelated FK506‐binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin‐negative (ppiB null) mutant and was shown to be the trigger factor (TigBS). N‐terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246° on the genetic map upstream of the clpX and lonA, B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS‐His proteins share the same substrate specificity and catalytic activity (kcat/Km of 1.5 μM−1 s−1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor.
Molecular Cloning, Sequence Analysis and Functional Characterization of the Gene Kdsa, Encoding 3‐Deoxy‐D‐Manno‐2‐Octulosonate‐8‐Phosphate Synthase of Chlamydia Psittaci 6BC
Authors: Brabetz Werner , Brade Helmut
Published: July 2004

The kdsA gene encoding 3‐deoxy‐D‐manno‐2‐octulosonate‐8‐phosphate (Kdo‐8‐P) synthase of Chlamydia psittaci 6BC was cloned by complementing the temperature‐sensitive kdsA mutant Salmonella enterica serovar Typhimurium AG701150. The sequence analysis of a recombinant DNA fragment revealed an open reading frame of 807 nucleotides which codes for a polypeptide of 269 amino acids with a high degree of similarity to known KdsA proteins. In addition, lignments of Kdo‐8‐P synthases with bacterial and fungal 3‐deoxy‐D‐arabino‐2‐heptulosonate‐7‐phosphate (Dha‐7‐P) synthases suggested that both classes of enzymes are structurally related and may belong to a family of 2‐keto‐3‐deoxy‐aldonic acid synthases. The chlamydial protein was overexpressed and functionally characterized in vitro to synthesize Kdo‐8‐P from D‐arabinose 5‐phosphate and phosphoenolpyruvate. A chlamydial DNA region upstream of the gene exhibiting similarities to the consensus sequence of σ70 promoters of Escherichia coli was responsible for the heterologous expression of kdsA.
Model of the pH‐Dependence of the Concentrations of Complexes Involving Metabolites, Haemoglobin and Magnesium Ions in the Human Erythrocyte
Authors: Mulquiney Peter J. , Kuchel Philip W.
Published: July 2004

The rate of glucose consumption and the concentrations of glycolytic intermediates in human erythrocytes have long been known to be pH sensitive. Despite the extensive literature on modelling erythrocyte metabolism, no model developed so far can adequately describe all of these pH‐dependent changes. None of these models have included all the significant association reactions between metabolites, Hb and Mg2+ that will influence metabolism. As part of a larger enterprise to develop a detailed model of erythrocyte glycolysis, we present a sub‐model which predicts, as a function of pH and oxygenation state, the concentrations of free and Mg2+‐bound metabolites that are substrates, co‐factors and effectors of glycolysis. This model shows that pH changes around physiological values can cause large changes in the distribution of metabolites between free, bound and Mg2+‐complexed forms, based on binding interactions alone; in oxygenated cells, at pH 7.2–7.6, many glycolytic intermediates undergo changes in concentration of 50–100%. The model also predicts intracellular concentrations of free Mg2+ in erythrocytes to be 0.4 mM and 0.64 mM in oxygenated and deoxygenated cells, respectively, assuming a total magnesium concentration of 3 mM (≈88% of the total magnesium usually found in erythrocytes). This is in close agreement with the values found by Flatman [Flatman, P. W. (1980)J. Physiol. 300, 19–30] and the finding by Flatman and Lew [Flatman, P. & Lew, V. L. (1977)Nature 267, 360–362] that the main Mg2+ buffer systems bind ≈90% of Mg2+ in the cell.

Hexokinase has a high ‘flux control coefficient’ in human erythrocyte glycolysis, so the dependence of its rate on the pH and oxygenation state of haemoglobin is important. With a low oxygen tension and an intracellular pH of 7.34, the major inhibitor of its activity (2,3‐bisphosphoglycerate) is 85 % bound to either haemoglobin or Mg2+, and the maximum possible flux of substrate via it would be 2.05 mmol L erythrocytes−1 h−1. However, if the haemoglobin were saturated with oxygen, and the pH were 7.2, it was calculated that the maximum rate would be 1.48 mmol L erythrocytes−1 h−1; this is primarily due to a doubling of the free 2,3‐bisphosphoglycerate concentration. However, the full extent of the inhibition is counteracted because the concentration of the Mg2+‐2,3‐bisphosphoglycerate would be approximately doubled. Many other similar comparisons are possible with this new model, which highlights the complex network of interactions between haemoglobin, Mg2+, H+ and the metabolites as substrates and effectors of the glycolytic reactions.
Memory and Imprinting Effects in Multienzyme Complexes
Authors: Avilan Luisana , Gontero Brigitte , Lebreton Sandrine , Ricard Jacques
Published: July 2004

A bienzyme complex made up of phosphoribulokinase and glyceraldehyde‐3‐phosphate dehydrogenase has been isolated and purified from chloroplasts of Chlamydomonas reinhardtii. The complex contains four phosphoribulokinase and eight glyceraldehyde‐3‐phosphate dehydrogenase polypeptide chains. As phosphoribulokinase is dimeric and glyceraldehyde‐3‐phosphate dehydrogenase tetrameric, it is concluded that the complex comprises two phosphoribulokinase and two glyceraldehyde‐3‐phosphate dehydrogenase molecules. Its overall molecular mass is 460 kDa, which is in excellent agreement with its stoichiometry. Moreover, owing to the nature of the two enzymes, this complex must catalyse two non‐consecutive reactions.

The bienzyme complex tended to spontaneously dissociate into the free enzymes upon dilution. This dissociation process was considerably promoted by reducing agents such as dithiothreitol or reduced thioredoxin. The kinetics of the dissociation process induced by dithiothreitol or reduced thioredoxin were paralleled by an increase of activity of phosphoribulokinase.

The dissociation of the complex was reversible. If oxidized phosphoribulokinase and glyceraldehyde‐3‐phosphate dehydrogenase were mixed, a certain amount of the complex was formed. The reconstituted complex displayed properties that were indistinguishable from those of the native complex extracted from chloroplasts of Chlamydomonas reinhardtii. These results suggest that the concentration of the complex in vivo must vary depending on the light intensity.
The Catalytic Domain of Activated Collagenase I (MMP‐1) is Absolutely Required for Interaction with Its Specific Inhibitor, Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1)
Authors: Vallon Rüdiger , Müller Ralph , Moosmayer Dieter , Gerlach Elke , Angel Peter
Published: July 2004

Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases‐1 (rTIMP‐1) and wild‐type and mutant human collagenase type I (rMMP‐1) proteins in SF9 cells by the baculovirus expression system. Wild‐type MMP‐1, as well as the MMP‐1 mutant lacking the C‐terminal hemopexin‐like domain [des‐(248–450)‐MMP‐1], exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4‐aminophenylmercuric acetate. Enzyme activity of both proteins can be inhibited by addition of rTIMP. Deletion of the complete active‐site [des‐(161–228)‐MMP‐1] within the catalytic domain, or mutation of a single His residue of the Zn2+ binding domain (His199), generates stable forms of MMP‐1 proteins which are unable to digest collagen type I or β‐casein. In addition to communo precipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP‐1 to form complexes with its inhibitor. Only the activated and not the latent forms of wild‐type and C‐terminal mutant des‐(248–450)‐MMP‐1 proteins are able to form complexes with TIMP. Neither mutation of Hisl99, nor deletion mutants des‐(161–228)‐MMP‐1 and des‐(161–228/248–450)‐MMP‐1, interact with TIMP. This demonstrates that the C‐terminal hemopexin domain of MMP‐1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP‐1. In summary, we have shown that the integrity of the catalytic domain of MMP‐1 and its ability to bind Zn2+ is absolutely required for complex formation with TTMP‐1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP‐1.
Structural Investigation of the Lipopolysaccharide from Acinetobacter Haemolyticus Strain NCTC 10305 (ATCC 17906, DNA Group 4)
Authors: Vinogradov Evgeny V. , Müller‐Loennies Sven , Petersen Bent O. , Meshkov Sergey , Thomas‐Oates Jane E. , Holst Otto , Brade Helmut
Published: July 2004

The structure of the lipopolysaccharide (LPS) from Acinetobacter haemolyticus strain NCTC 10305 (DNA group 4) was elucidated by means of analytical chemistry, NMR spectroscopy and fast‐atom‐bombardment mass spectrometry. Several oligosaccharides were obtained after deacylation or successive de‐O‐acylation, dephosphorylation, reduction, and de‐N‐acylation of LPS. In the major fraction of the LPS, the core is attached to the lipid A through d‐glycero‐d‐talo‐2‐octulopyranosonic acid (Ko), whereas in a minor fraction (<20%) Ko is replaced by 3‐deoxy‐d‐manno‐octulopyranosonic acid (Kdo). The structures of the phosphorylated carbohydrate backbones of these LPS fractions are
image

with Dha = 3‐deoxy‐d‐lyxo‐2‐heptulosaric acid, Sug = sugar and is Ko in a major fraction and Kdo in a minor fraction. All sugar residues have the d‐configuration and are present in the pyranose form. Mass spectrometry of de‐O‐acylated LPS revealed the presence of an additional hexose residue in minor amounts, the position and nature of which could not be identified.
Reconstitution of T Cell Antigen Receptor‐Induced Erk2 Kinase Activation in Lck‐Negative JCaM1 Cells by Syk
Authors: Williams Scott , Couture Clément , Gilman Jennifer , Jascur Thomas , Deckert Marcel , Altman Amnon , Mustelin Tomas
Published: July 2004

The two related protein‐tyrosine kinases Syk and Zap are rapidly phosphorylated on tyrosine residues and enzymatically activated upon crosslinking of the T cell antigen receptor. We have previously reported that the activation of Syk is less dependent on the Src family kinase Lck than the activation of Zap. Here we report that overexpression of Syk in the Lck‐negative JCaM1 cells enabled the T cell antigen receptor/CD3 complex to induce a normal activation of the mitogen‐activated protein kinase (MAPK) pathway and expression of a nuclear factor of activated T cells reporter construct. In contrast, Zap and other protein‐tyrosine kinases were unable to reconstitute these signaling pathways when expressed at the same levels. In parallel, Syk was phosphorylated on tyrosine, while Zap was not. The Syk‐mediated T cell antigen receptor‐induced MAPK activation was detectable within 1 min of receptor stimulation and peaked at 3–5 min. The capacity of Syk to reconstitute the MAPK response required the catalytic activity of Syk, an intact autophosphorylation site (Y518 and Y519), both Src homology 2 domains and it was blocked by the inhibitory N17‐mutated dominant‐negative Ras construct. A Y341→F mutant of Syk, which is deficient in its interaction with phospholipase Cγ1 and Vav, was less efficient than wild‐type Syk. Our results suggest that Syk, in contrast to Zap, can transduce signals from the T cell antigen receptor independently of Lck.
Structures of Phenolic Glycolipids from Mycobacterium Kansasii
Authors: Watanabe Motoko , Aoyagi Yutaka , Ohta Akihiro , Minnikin David E.
Published: July 2004

From the lipid fraction of cells of Mycobacterium kansasii, four phenolic glycolipids K5, K6, K7 and K8, were isolated, in addition to three known phenolic glycolipids KI, KII and KIV. K5 was identified as a tetraglycosyl phenolic glycolipid whose sugar moiety was 2,6‐dideoxy‐4‐O‐methyl‐l‐α‐arabinohexopyranosyl(1→3)‐4‐O‐propionyl‐2‐O‐methyl‐l‐α‐fucopyranosyl(1→3)‐2‐O‐methyl‐l‐α‐rhamnopyranosyl‐(1→3)‐2,4‐di‐O‐methyl‐l‐α‐rhamnopyranosyl(1→) and phenolic glycolipid K6 as deacetyl‐KI. Glycolipids K7 and K8 were triglycosyl phenolic glycolipids having the sugar moieties of 2‐O‐methyl‐l‐α‐fucopyranosyl(1→3)‐2‐O‐methyl‐l‐α‐rhamnopyranosyl(1→3)‐2,4‐di‐O‐methyl‐l‐α‐rhamnopyranosyl(1→) and 2‐O‐methyl‐l‐α‐fucopyranosyl(1→3)‐2‐O‐methyl‐l‐α‐rhamnopyranosyl(1→3)‐2‐O‐methyl‐l‐α‐rhamnopyranosyl(1→), respectively. Phenolic glycolipids K6 and K7 have been referred to as controlled degradation products of phenolic glycolipid KI previously. Also isolated was 5‐mycolyl‐β‐arabinofuranosyl(1→2)‐5‐mycolyl‐α‐arabinofurnosyl(1→1′)‐glycerol, an analogue of glycolipid ai, originally isolated from Mycobacterium avium‐Mycobacterium intracellulare complex, having mycolic acids of M. kansasii.
Transgenic Mice Transcribing the Human H1° Histone Gene Exhibit a Normal Phenotype
Authors: Tönjes Ralf R. , Paul Dieter , Doenecke Detlef
Published: July 2004

The linker histone H1° accumulates in terminally differentiating cells and replaces other members of the H1 histone family, even in the absence of cell division. To study the role of H1°in vivo, we have created two lines of transgenic mice with either the human H1° promoter (HH minigene) or the mouse metallothionein I promoter (MH minigene) upstream of the human H1° gene. Mice bearing the minigenes HH or MH overexpress human H1° mRNA at 10–20‐fold higher levels than in normal mice in a constitutive or metal‐inducible manner. In contrast to this increase in mRNA content, which was studied in liver, kidney and brain, no significant changes in the relative proportions of the H1 protein subtypes, including H1° were observed. Transgenic mice exhibited normal anatomic phenotypes, growth rates and reproduction rates. Thus, our results suggest a posttranscriptional and/or translational mechanism that compensates the unbalanced linker‐histone expression in different tissues.
Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells
Authors: Lerche Carole , Fautrel Alain , Shaw Peter M. , Glaise Denise , Ballet Francois , Guillouzo Andre , Corcos Laurent
Published: July 2004

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P‐450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase α (GSTα), aldehyde dehydrogenase and epoxide hydrolase in co‐cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O‐deethylation activity was well maintained and ethoxyresorufin O‐deethylation activity gradually decreased during co‐culture time. In addition, we showed that phenobarbital and 3‐methylcholanthrene treatments resulted in a significant increase of these activities.

Two general patterns of accumulation of liver‐specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GSTa, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6‐fold) and GSTa (3.9‐fold) mRNA expression in 3‐methyl cholanthrene‐treated co‐cultures and induction of CYP2B1/2 (19‐fold), CYP2C6 (10‐fold), CYP3A1/2 (11.2‐fold), GSTα (9‐fold), aldehyde dehydrogenase (6‐fold) and epoxide hydrolase (5‐fold) mRNA expression in phenobarbital‐treated co‐cultures. Furthermore, we demonstrated that liver‐specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co‐culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells.

Finally, to investigate whether co‐cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co‐culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163‐bp DNA sequence element reported to confer phenobarbital responsiveness. A 2–3‐fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital‐treated co‐cultures transfected with the phenobarbital‐responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments.

Our results demonstrate that the co‐culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.
Mutation of Amino Acids 39–44 of Human CD14 Abrogates Binding of Lipopolysaccharide and Escherichia coli
Authors: Stelter Felix , Bernheiden Martin , Menzel René , Jack Robert S. , Witt Sabine , Fan Xiaolong , Pfister Martin , Schütt Christine
Published: July 2004

As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non‐specific host defense mechanisms against gram‐negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N‐terminal 252 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti‐CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. 4 of 21 expressed CD14 mutants, ([Ala9–Ala13]CD14, [Ala39–Ala41, Ala43, Ala44]CD14, [Ala51–Ala55]CD14 and [Ala57, Ala59, Ala61–Ala63]CD14), are not recognized by anti‐CD14 mAbs that interfere with the binding of LPS to human monocytes. However, only [Ala39–Ala41, Ala43, Ala44lCD14 is unable to react with fluorescein‐isothiocyanate‐labeled LPS or with FITC‐labeled E. coli (055:B5). In addition, [Ala39–Ala4t. Ala43, Ala44]CD14 does not mediate LPS (E. coli 055:B5; 10 ng/ml)‐induced translocation of nuclear factor κB in CHO‐cell transfectants. The results indicate that the region between amino acids 39 and 44 forms an essential part of the LPS‐binding site of human CD14.