bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrous oxide diffusion into tracheal tube cuffs: comparison of five different tracheal tube cuffs
Authors: Dullenkopf A. , Gerber A. C. , Weiss M.
Published: September 2004

Background:  The aim of this study was to investigate cuff compliance and cuff pressure during nitrous oxide exposure in the recently introduced Microcuff tracheal tube with a polyurethane cuff (Microcuff GmbH, Weinheim, Germany), and to compare it to conventional tracheal tubes with PVC cuffs.

Methods:  In an in vitro set up, five cuffed tracheal tubes (ID 7.0 mm) from different manufacturers (Microcuff HVLP, Portex Profile Soft Seal, Mallinckrodt HiLo, Rüsch Super Safety Clear and Sheridan CF) were studied. Pressure–volume curves were assessed and changes of cuff pressure during exposure to nitrous oxide (for 60 min; 66% N2O in oxygen) were recorded without and with restriction of the cuff in a trachea model. Each experiment was performed four times using two exemplars of each tube twice. Sixty‐minute values of the Microcuff group were compared with the other groups using the Mann– Whitney test (P < 0.05).

Results:  The Microcuff polyurethane cuff demonstrated an intermediate cuff compliance but the highest cuff pressure increase during nitrous oxide exposure under unrestricted conditions. When inflated within the artificial trachea its cuff compliance became the highest of all tested tracheal tubes. However, exposure to N2O, again led to a rapid increase in cuff pressure.

Conclusion:  The ultra‐thin polyurethane tube cuff demonstrated higher permeability for nitrous oxide than conventional PVC cuffs and led to a rapid cuff pressure increase when exposed to N2O. Routinely checking of cuff‐pressure or filling the cuff with nitrous oxide are more important than the brand of tube used.
Clinical application of the pO2–pCO2 diagram
Authors: Paulev P.‐E. , Siggaard‐Andersen O.
Published: September 2004

Based on the classic, linear blood gas diagram a logarithmic blood gas map was constructed. The scales were extended by the use of logarithmic axes in order to allow for high patient values. Patients with lung disorders often have high arterial carbon dioxide tensions, and patients on supplementary oxygen typically respond with high oxygen tensions off the scale of the classic diagram. Two case histories illustrate the clinical application of the logarithmic blood gas map. Variables from the two patients were measured by the use of blood gas analysis equipment. Measured and calculated values are tabulated. The calculations were performed using the oxygen status algorithm. When interpreting the graph for a given patient it is recommended first to observe the location of the marker for the partial pressure of oxygen in inspired, humidified air (I) to see whether the patient is breathing atmospheric air or air with supplementary oxygen. Then observe the location of the arterial point (a) to see whether hypoxemia or hypercapnia appears to be the primary disturbance. Finally observe the alveolo‐arterial oxygen tension difference to estimate the degree of veno‐arterial shunting. If the mixed venous point (v) is available, then observe the value of the mixed venous oxygen tension. This is the most important indicator of global tissue hypoxia.
Effects of nitrous oxide on baroreflex gain and heart rate variability
Authors: Tanaka M. , Nishikawa T.
Published: September 2004

Background:  Spontaneous baroreflex method allows continuous assessment of cardiovagal reflex function within resting blood pressure, but effects of nitrous oxide, per se, on the spontaneous baroreflex response remain unknown. This study was designed to determine the effects of nitrous oxide on spontaneous baroreflex gain and heart rate variability assessed by power spectral analysis in humans.

Methods:  Electrocardiogram and non‐invasive blood pressure were monitored in 12 healthy volunteers before and during a 15‐min inhalation of 67% nitrous oxide in oxygen, while spontaneous respiration was maintained. Least‐square regression analysis relating R‐R interval and systolic blood pressure was performed to obtain spontaneous baroreflex gains. Heart rate variability was analyzed using fast Fourier transformation.

Results:  Nitrous oxide did not significantly alter spontaneous baroreflex gains, which correlated well with high‐frequency power (0.15–0.4 Hz) of heart rate variability before and during nitrous oxide inhalation.

Conclusion:  Our results indicate that (a) cardiovagal reflex response is not affected by nitrous oxide, per se, and (b) spontaneous baroreflex responses closely reflect beat‐to‐beat dynamic modulation of the cardiac cycle by the parasympathetic nervous system during inhalation of 67% nitrous oxide.
Predictable reduction of intracranial hypertension with hypertonic saline hydroxyethyl starch: a prospective clinical trial in critically ill patients with subarachnoid haemorrhage
Authors: Bentsen G. , Breivik H. , Lundar T. , Stubhaug A.
Published: September 2004

Background:  After head trauma, hypertonic saline lowers intracranial pressure (ICP) and preserves or increases cerebral perfusion pressure (CPP). Hypertonic saline has not been studied in patients with increased ICP due to subarachnoid haemorrhage (SAH). The aim of this study was to evaluate the effects on elevated ICP and on CPP in patients critically ill from SAH.

Methods:  Critically ill SAH‐patients needing urgent treatment for an elevated ICP, but otherwise stable, were included in this study. We infused 7.2% saline in 6% hydroxyethyl starch (HyperHAES® Fresenius Kabi AG, Bad Homburg v.d.h., Germany) 2 ml kg−1 during 20 min in 10 episodes of ICP > 20 mmHg in seven patients with SAH. Our primary outcome variables were changes in ICP and CPP during and for 3 h after this infusion.

Results:  All interventions resulted in decreased ICP and elevation of CPP. The mean value for maximum ICP decrease in percent of baseline was 58% (range 43–83%, P = 0.002), which occurred at mean 40 min (range 25–90 min) after start of infusion. The mean percent peak increase in CPP was 26% (range 16–32%, P = 0.002). After 210 min, ICP was 35% lower than baseline (range 19–39%, P = 0.008). Serum sodium increase was mean 6.6 mmol l−1 (range 5–9 mmol l−1) 30 min after start of infusion.

Conclusions:  7.2% saline in 6% hydroxyethyl starch is an effective and safe therapy for intracranial hypertension after SAH. We demonstrate that an infusion of 2 ml kg−1 during 20 min has a predictable and clinically significant beneficial effect on ICP and CPP. The effect was still present 3 h after end of infusion. Rebound ICP‐increase was not observed within 3 h.
Comparison of BIS and AAI as measures of anaesthetic drug effect during desflurane–remifentanil anaesthesia
Authors: Kreuer S. , Bruhn J. , Larsen R. , Bauer C. , Wilhelm W.
Published: September 2004

Background:  Autoregressive modelling with exogenous input of the middle‐latency auditory evoked potential has been developed for monitoring of anaesthetic depth. This study was designed to investigate the dose–response relationship between endtidal desflurane concentrations and the Alaris Autoregressive Index (AAI, Alaris Medical, Hampshire, UK, version 1.4) or the bispectral index (Aspect Medical Systems, Newton, MA, USA, version XP).

Methods:  Twenty‐one patients scheduled for radical prostatectomy were investigated. After premedication and induction of anaesthesia with propofol und remifentanil all patients received atracurium and a remifentanil background infusion at a constant rate of 0.1 µg kg−1 min−1. During dissection of the prostate, desflurane endtidal concentrations were varied between 3 and 9 vol%. Both AAI and BIS were determined and compared with the respective endtidal desflurane concentration.

Results:  None of the patients showed a significant change of AAI values while changing the desflurane concentrations between 3 and 9 vol%. The dose–response of BIS values and desflurane concentrations was not uniform: two patients showed increasing BIS values with increasing desflurane concentrations, while in three patients BIS values remained unchanged. In 16 patients decreasing BIS values adequately reflected an increase in desflurane concentrations.

Conclusion:  Changes of desflurane concentrations during deep anaesthesia were adequately displayed only in 16 of 21 cases by BIS but in none of the cases by AAI monitoring.
Total spinal anaesthesia as a complication of local anaesthetic test‐dose administration through an epidural catheter
Authors: Steffek M. , Owczuk R. , Szlyk‐Augustyn M. , Lasinska‐Kowara M. , Wujtewicz M.
Published: September 2004

We describe a case of total spinal anaesthesia, which occurred after a 3‐ml lignocaine (20 mg ml−1) test dose was administered through an epidural catheter in a 79‐year‐old patient scheduled for gastrectomy under combined general and epidural anaesthesia.

The surgery was postponed, and the patient required admission to the intensive therapy unit. Spinal MRI from the total spinal cord did not reveal any pathology. During the next 24 h the patient recovered and after 11 days was successfully operated on under general anaesthesia. No late complications followed. We presume that during placement, the epidural catheter had migrated to the spinal canal as a result of technical difficulties.

Although controversial, we consider that administering a standard test dose of local anaesthetic via an epidural catheter is recommended, especially in high‐risk patients and when epidural space identification or catheter placement poses technical difficulties. A test dose of local anaesthetic does not fully prevent complications.
Postoperative cognitive dysfunction: true deterioration versus random variation
Authors: Rasmussen L. S. , Siersma V. D. ,   the ispocd group
Published: September 2004

Background:  Postoperative cognitive dysfunction (POCD) is a common complication, especially in the elderly. The aim of this study was to describe how variability in neuropsychological testing could lead to the detection of cognitive improvement and poor consistency of POCD between postoperative test sessions.

Methods:  In four published studies performed by the ISPOCD group, we included 2536 patients and 359 healthy controls. Cognitive function was assessed using neuropsychological tests preoperatively and at 7 days and 3 months thereafter, comparing the changes between those at baseline with those after surgery. Postoperative cognitive dysfunction was defined as a Z score greater than 2, and we also defined a corresponding improvement as a Z score less than −2. Consistency of POCD between postoperative test sessions was analyzed and we also assessed test–retest variability using data from healthy control subjects.

Results:  Improvement in cognitive function was found in 4.2–8.7% of patients after 1 week and in 5.0–7.8% after 3 months. The ratio between incidence of dysfunction and improvement varied in patients between 3.3 and 6.2 early after major surgery. Of those patients who displayed POCD at the 3‐month test, 30–48% also had POCD at the previous test at 1 week. The test–retest reliability was between 0.56 and 0.90, except for the error score in Concept Shifting Test, where the values were 0.20 and 0.37.

Conclusion:  Variability in neuropsychological test data contributes to a low consistency between postoperative test sessions but it does not explain the detection of cognitive dysfunction after major surgery.
Properties of Isolated Domains of the Elongation Factor Tu from Thermus Thermophilus HB8
Authors: Nock Steffen , Grillenbeck Norbert , Ahmadian Mohammad Reza , Ribeiro Sofia , Kreutzer Roland , Sprinzl Mathias
Published: September 2004

The relative contributions of the three domains of elongation factor Tu (EF‐Tu) to the factor's function and thermal stability were established by dissecting the domains apart with recombination techniques. Domain I (EF‐Tu1), domains I/II (EF‐TuI/II) and domain III (EF‐TuIII) of the EF‐Tu from Thermus thermophilus HB8 comprising the amino acids 1–211, 1–312 and 317–405, respectively, were overproduced in Escherichia coli and purified. A polypeptide consisting of domain II and III (EF‐TuII/III) was prepared by limited proteolysis of native EF‐Tu with V8 protease from Staphylococcus aureus [Peter, M. E., Reiser, C. O. A., Schirmer, N. K., Kiefhaber, T., Ott, G., Grillenbeck, N. W. & Sprinzl, M. (1990) Nucleic Acids Res. 18, 6889–6893]. As determined by circular dichroism spectrometry, the isolated domains have the secondary structure elements found in the native EF‐Tu. GTP and GDP binding as well as GTPase activity are maintained by the EF‐Tu1 and EF‐TuI/II; however, the rate of GDP dissociation from EF‐Tu1· GDP and EF‐TuI/II. GDP complex is increased as compared to native EF‐Tu · GDP, reflecting a constraint imposed by domain III on the ability to release the nucleotide from its binding pocket located in domain I. A weak interaction of Tyr‐tRNATyr with the EF‐Tu1· GTP suggests that domain I provides a part of the structure interacting with aminoacyl‐tRNA. The domain III is capable of regulating the rate of GTPase in EF‐Tu, since the polypeptide consisting only of domains I/II has a 39‐fold higher intrinsic GTPase compared to the native EF‐Tu. No in vitro poly(U)‐dependent poly(Phe) synthesis was detectable with a mixture of equimolar amounts of domains I/II and domain III, demonstrating the necessity of covalent linkage between the domains of EF‐Tu for polypeptide synthesis. In contrast to native EF‐Tu and EF‐TuII/III, EF‐Tu1 and, to a lesser extent the polypeptide consisting of domains I/II, are unstable at elevated temperatures. This indicates that domains II/III strongly contribute to the thermal stability of this T. thermophilus EF‐Tu. Deletion of amino acid residues 181–190 from domain I of T. thermophilus EF‐Tu decreases the thermostability to that of EF‐Tu from E. coli, which does not have these residues. Interdomain interactions must be important for the stabilisation of the structure of domain I, since isolated T. thermophilus EF‐Tu1 is thermolabile despite the presence of the 181–190 loop. Nucleotide exchange factor Ts (EF‐Ts), which promotes the dissociation of the nucleotide from native EF‐Tu, does not accelerate the nucleotide exchange from EF‐TuI· GDP and EF‐TuI/II· GDP polypeptides.
Identification of Two Sites in Gelsolin with Different Sensitivities to Adenine Nucleotides
Authors: Laham Lorraine E. , Way Michael , Yin Helen L. , Janmey Paul A.
Published: September 2004

The affinity of monomeric actin for several actin‐binding proteins, including gelsolin, depends on adenine nucleotides. Gelsolin binds faster and with higher affinity to ADP‐actin than to ATP‐actin. Here, we show that the C‐terminal actin‐binding domain of gelsolin, which is required for filament nucleating activity but not for filament severing activity, contains the site that distinguishes between ATP‐actin and ADP‐actin monomers.

In contrast, actin binding to the N‐terminal half of gelsolin depends on solution ATP concentrations, but not on the nucleotide (ATP or ADP) tightly bound in the cleft of the actin monomer. Binding is stronger in the absence of free nucleotide or in the presence of 0.5 mM ADP than in solutions containing 0.5 mM ATP. Complexes formed using different nucleotide concentrations differ in their filament‐severing activities as well as in their abilities to increase the fluorescence of 4‐chloro‐7‐nitrobenzeno‐2‐oxa‐1,3‐diazole‐labeled actin monomers. These results suggest that, at physiologic concentrations of nucleotides, both free and actin‐bound ATP may affect the binding of actin to its accessory proteins and that gelsolin, actin, or the gelsolin‐actin complex, contains a low‐affinity nucleotide‐binding site.
Isolation of a Novel cDNA that Encodes a Protein Localized to the Pre‐Acrosome Region of Spermatids
Authors: Aoto Hiroshi , Tsuchida Junji , Nishina Yukio , Nishimune Yoshitake , Asano Akira , Tajima Shoji
Published: September 2004

We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5‐azacytidine, a potent demethylating reagent. The amount of transcript increased with 5‐azacytidine treatment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841‐nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107 254 Da. Specific antibodies raised against the fusion protein including glutathione S‐transferase revealed an approximately 130‐kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western‐blot analysis. The protein was localized to the pre‐acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.
Activation of two Isoforms of Mitogen‐Activated Protein Kinase Kinase in Response to Epidermal Growth Factor and Nerve Growth Factor
Authors: Moriguchi Tetsuo , Gotoh Yukiko , Nishida Eisuke
Published: September 2004

Mitogen‐activated protein kinase kinase (MAPKK) is a dual‐specificity protein kinase which phosphorylates and activates mitogen‐activated protein kinase (MAPK). cDNAs encoding two isoforms of MAPKK, MAPKK1 and MAPKK2 (also known as MEK1 and MEK2), have been cloned in mammalian cells. To analyze the characteristics of MAPKK1 and MAPKK2 individually, we have produced specific anti‐MAPKK serum against each isoform. MAPKK1 and MAPKK2 have apparent molecular masses of 45 kDa and 47 kDa, respectively, on SDS/polyacrylamide gel electrophoresis. In mouse tissues, MAPKK1 was highly enriched in brain, while MAPKK2 was present relatively evenly. In rat fibroblastic 3Y1 cells, epidermal growth factor (EOF) treatment induced activation of both MAPKK1 and MAPKK2.

Immunoprecipitation experiments have shown that the time courses of activation and deactivation of both isoforms of MAPKK were superimposed. In PC12 cells, both MAPKK1 and MAPKK2 were activated in response to nerve growth factor (NGF) as well as EOF, and the time courses of activation and deactivation of both isoforms were indistinguishable from each other in the NGF‐stimulated cells and also in the EGF‐stimulated cells. Furthermore, localization of both MAPKK1 and MAPKK2 in the cytoplasm was unchanged in response to EGF and NGF. Thus, the same or quite similar mechanisms may operate in the regulation of the activation and deactivation of two isoforms of MAPKK, and both kinases might have redundant functions when expressed in the same cell.
Regulation of the Expression of Cathepsin B in Sarcophaga Peregrina (Flesh Fly) at the Translational Level During Metamorphosis
Authors: Yano Tamaki , Takahashi Noboru , Kurata Shoichiro , Natori Shunji
Published: September 2004

Previously, we reported that cathepsin B of pupal hemocytes participates in dissociation of the fat body of Sarcophaga larvae at metamorphosis. In this study, we demonstrated that the amount of cathepsin B in hemocytes was controlled at the translational level, i.e. larval hemocytes stored a significant amount of untranslated cathepsin B mRNA. When the larvae pupated, translation of the mRNA commenced, resulting in accumulation of cathepsin B. Our results indicate that the 3′‐untransJated region (3′‐UTR) of this mRNA is necessary for the repression of its translation in a rabbit reticulocyte lysate. We detected the 3′‐UTR binding protein in the lysate of larval hemocytes, but not in the pupal hemocytes. The role of this protein in the translation of cathepsin B mRNA is discussed.
A Specific Template‐Assembled Peptidic Agonist for the Angiotensin II Receptor Subtype 2 (AT2) and its Effect on Inferior Olivary Neurones
Authors: Grouzmann Eric , Felix Dominik , Imboden Hans , Razaname Alain , Mutter Manfred
Published: September 2004

We synthesized a molecule composed of two angiotensin II 4–8 pentapeptide fragments attached to a carrier molecule (TA), according to the template‐assembled synthetic proteins concept. This molecule was investigated for receptor binding on angiotensin type‐1 and type‐2 receptors (AT1 and AT2) and its biological activity was determined by iontophoretic experiments on neurones of the inferior olive (ION) that express only AT2 receptors. TA binds exclusively to the AT2 receptor and mediates an agonistic angiotensin‐II effect on the ION. TA is the first agonist available to study the direct stimulation of AT2 receptors.
Phosphorylation of Calmodulin by Plasma‐Membrane‐Associated Protein Kinase(s)
Authors: BengurÍA Alberto , Soriano Montserrat , Joyal John L. , Sacks David B. , Villalobo Antonio
Published: September 2004

Plasma‐membrane‐associated protein kinase(s) from normal rat liver phosphorylates exogenous bovine brain calmodulin in the absence of Ca2+ and in the presence of histone or poly(l‐lysine). Maximum levels of calmodulin phosphorylation are obtained at a poly (l‐lysine)/calmodulin molar ratio of 0.4. Phosphoamino acid analysis revealed that calmodulin is phosphorylated on serine, threonine and tyrosine residues. Endogenous plasma‐membrane‐associated calmodulin was also phosphorylated by plasma‐membrane‐associated protein kinase(s) in the absence of added cationic protein or polypeptide. The identity of endogenous phosphocalmodulin was confirmed by immunoprecipitation with a specific anti‐calmodulin monoclonal antibody. Ehrlich ascites tumor cell plasma membranes do not contain endogenous calmodulin. However, membrane‐associated protein kinase(s) from these tumor cells phosphorylates bovine brain calmodulin in the presence of poly(l‐lysine). These data demonstrate that phosphocalmodulin is present in liver plasma membranes and suggest that this post‐translational modification could have a physiological role in this location.
Characterization of Two Genes Coding for a Similar Four‐Cysteine Motif of the Amino‐Terminal Propeptide of a Sea Urchin Fibrillar Collagen
Authors: Exposito Jean‐Yves , Boute Nicolas , Deleage Gilbert , Garrone Robert
Published: September 2004

We report the characterization of the 5′ region of the gene coding for the 2α fibrillar collagen chain of the sea urchin Paracentrotus lividus. This sequence analysis identified the intron/exon organization of the region of the gene coding for the signal peptide, the cysteine‐rich domain and the 12 repeats of the four‐cysteine module of the unusually long amino‐propeptide. This still unknown four‐cysteine motif is generally encoded by one exon, which confirms that the distinct amino‐propeptide structures of the fibrillar collagens arise from the shuffling of several exon‐encoding modules. Moreover, Southern‐blot analysis of the sea urchin genome and sequencing of selected genomic clones allowed us to demonstrate that several sea urchin genes could potentially code for the four‐cysteine module. Curiously, one of these genes lacks the exons coding for four repeats of this motif while, in another gene, the same exons are submitted to an alternative splicing event.
The Signal Transduction Pathway of Erythropoietin Involves Three Forms of Mitogen‐Activated Protein (MAP) Kinase in UT7 Erythroleukemia Cells
Authors: Gobert Stephanie , Duprez Veronique , Lacombe Catherine , Gisselbrecht Sylvie , Mayeux Patrick
Published: September 2004

The survival and proliferation of the UT‐7 human leukemic cell line is strictly dependent on the presence of either interleukin 3, granulocyte‐macrophage colony‐stimulating factor or erythropoietin. In these cells, erythropoietin stimulation led to the rapid phosphorylation of several proteins including the erythropoietin receptor and proteins with molecular masses around 45 kDa which could be rnitogen‐activated protein (MAP) kinases. Separation of cytosol from resting or erythropoietin‐stimulated UT‐7 cells by anion‐exchange chromatography revealed two peaks of myelin basic protein kinase activity. The kinase activity of the first peak was independent of erythropoietin treatment of the cells and corresponded to an unidentified 50‐kDa kinase, whereas the second peak was only present in erythropoietin‐stimulated cells and corresponded to three forms of MAP kinases with molecular masses of 45, 44 and 42 kDa. The three forms were separated by hydrophobic chromatography and were shown to be activated in erythropoietin‐stimulated cells. The 44‐kDa and 42‐kDa forms corresponded to extracellular signal‐regulated kinase (ERK)‐1 and ERK‐2, respectively. Evidence was obtained showing that the 45‐kDa form is not a shifted form of ERK‐1 but corresponded to a less well defined form of MAP kinase which may be the previously described ERK‐4. MAP kinase activation was detected after 1 min erythropoietin stimulation and remained detectable after more than 1 hour. A role for MAP kinase activation in erythropoietin‐stimulated cell proliferation was suggested by the simultaneous inhibition of erythropoietin‐induced MAP kinase stimulation and cell proliferation. The potential activator of MAP kinase, RAF‐1, was hyperphos‐phorylated in erythropoietin‐stimulated cells and its autophosphorylation activity was strongly increased. The protein adaptor Shc was heavily phosphorylated in UT‐7 erythropoietin‐stimulated cells and associated strongly with a unidentified 145‐kDa protein. However, She bound poorly to the activated erythropoietin receptor and most She proteins were cytosolic in both unstimulated and erythropoietin‐stimulated cells. In contrast, Grb2 associated efficiently with the activated erythropoietin receptor and a significant part of Grb2 was associated to a particulate subcellular fraction upon erythropoietin stimulation.
Isoproterenol Inhibits Insulin‐Stimulated Tyrosine Phosphorylation of the Insulin Receptor Without Increasing its Serine/Threonine Phosphorylation
Authors: Issad Tarik , Combettes Murielle , Ferre Pascal
Published: September 2004

The effect of a β‐adrenergic agonist (isoproterenol) on the tyrosine kinase activity of the insulin receptor was studied in intact adipocytes. Isoproterenol treatment rapidly (5 min) inhibited the insulin‐induced autophosphorylation of the insulin receptor on tyrosine residues in intact adipocytes. The effect of insulin on the phosphorylation of cellular proteins on tyrosine residues was also inhibited by isoproterenol. In order to understand the mechanism responsible for this inhibition, two‐dimensional phosphopeptide mapping of the insulin receptor was performed. The pattern of phosphorylation of the insulin receptor in freshly isolated adipocytes showed marked differences from that previously observed in cultured cells overexpressing insulin receptors. These differences include a larger proportion of receptors being phosphorylated on the three tyrosines from the kinase domain and no apparent phosphorylation of the two tyrosines close to the C‐terminus after insulin stimulation. Isoproterenol markedly inhibited the effect of insulin on the phosphorylation of the three tyrosines from the kinase domain. However, this inhibition was not associated with an increase in the phosphorylation of serine/threonine peptides. Thus, this direct analysis of insulin receptor phosphorylation sites in intact adipocytes does no support the idea that β‐adrenegic agents inhibit the tyrosine kinase activity of the receptor through a serine/threonine phosphorylation‐dependent mechanism.
Structure and Expression Analysis of the gdcsPA and gdcsPB Genes Encoding Two P‐Isoproteins of the Glycine‐Cleavage System from Flaveria Pringlei
Authors: Bauwe Hermann , Chu Chen‐cai , Kopriva Stanislav , Nan Qu
Published: September 2004

In Flaveria pringlei, a C3 plant, P protein of the glycine‐cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P‐protein genes can be subdivided into two parts. In both cases the N‐terminal region consists of one very long exon followed by a long intron. In contrast, the C‐terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine‐zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse‐transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter‐β‐glucuronidase fusions in transgenic tobacco suggests that far‐upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.
Immunohistochemical and Biochemical Analyses of 20000–25000‐Year‐Old Fossil Cartilage
Authors: Franc Suzanne , Marzin Eric , Boutillon Marguerite‐Marie , Lafont Raymond , LechÊne De La Porte Paulette , Herbage Daniel
Published: September 2004

A cracked, irregular pellicle adhering to fossilized bone excavated from the Enléne cave (Ariége) and estimated to date from 20000–25000 years BP was examined to verify its cartilaginous nature, suggested previously on the basis of optical and electron microscopic investigations. Immunolabeling of the organic component revealed the presence of type II and IX collagens, associated with residual glycosaminoglycans, in the external zone of the pellicle. The cartilaginous nature of the pellicle was also demonstrated by biochemical identification of type II collagen as the major protein in the demineralized sample: the amino acid compositions of insoluble and soluble fractions were similar to that of pure type II collagen; cyanogen‐bromide‐generated peptides, prepared after reduction of the sample, had an electrophoretic pattern similar to that of cyanogen bromide peptides derived from type II collagen. The amino acid sequences of four tryptic peptides were identical to the corresponding human type II sequences. It was impossible to isolate intact α chains. All of the solubilized fractions were composed of a wide range of low‐molecular‐mass peptides demonstrating significant degradation of the collagen molecules that was not reflected in the well‐preserved fibrillar structure observed at the ultrastructural level. The mineral fraction, characterized by X‐ray diffraction, consisted of apatite (as in sub‐chondral bone) associated with contaminating poorly crystallized components originating from the cave sediment. Energy dispersive spectrometry showed that the cartilaginous zone contained three times less phosphorus and calcium than the underlying bone. These results confirm the cartilaginous nature of the sample and the preservation of tissue‐specific components, and suggest that the process of fossilization is closely related to a mechanism of phosphatization.
Reversible Dissociation and Unfolding of Dimeric Creatine Kinase Isoenzyme MM in Guanidine Hydrochloride and Urea
Authors: Couthon Fabienne , Clottes Eric , Ebel Christine , Vial Christian
Published: September 2004

The unfolding of dimeric cytoplasmic creatine kinase (MM) by guanidine hydrochloride and by urea has been investigated using activity measurements, far‐ultraviolet circular dichroism, sedimentation velocity and fluorescence energy transfer experiments to monitor global structural changes. Intrinsic (cysteine and tryptophan residues) and extrinsic probes (1‐anilinonaphthalene‐8‐sulfonate) were also used. The reversibility of the unfolding was checked by monitoring activity and tryptophan fluorescence. The unfolding of creatine kinase in guanidine hydrochloride is a reversible multistep process, as suggested by the non‐coincidence of denaturation curves at equilibrium. Inactivation of the dimer precedes its dissociation into two monomers and an intermediate state was identified during the unfolding of the monomer. This intermediate state is characterized by a relatively high degree of secondary structure (as shown by far‐ultraviolet circular dichroism), of compactness (as shown by fluorescence energy transfer measurements and sedimentation experiments), a fluctuating tertiary structure (as shown by near‐ultraviolet circular dichroism) and a strong affinity for anilinonaphthalene sulfonate (as demonstrated by fluorescence). These results clearly indicate that the intermediate state detected possesses some of the properties of a molten globule. In urea, the unfolding pathway is reversible but differs from that observed in guanidine hydrochloride. Indeed inactivation, dissociation and loss of tertiary structure are coincident but the ellipticity curve is slightly shifted to a higher urea concentration. The dimer is dissociated into two expanded monomers possessing some secondary structure which is progressively lost at a higher urea concentration (6.4 M).

These results show that guanidine hydrochloride is approximately six times more effective than urea for inactivation and dissociation, underlining the fact that electrostatic interactions are very important in the stabilization of the active site and of the dimeric state.
Identification of a Three‐Amino‐Acid Region in G Protein γ1 as a Determinant of Selective βγ Heterodimerization
Authors: Meister Michael , Dietrich Alexander , Gierschik Peter
Published: September 2004

Guanine‐nucleotide‐binding protein β and γ subunits belong to large protein families encompassing at least five and ten members, respectively, from mammalian cells. The formation of stable βγ heterodimers is a selective process determined by the primary sequences of both the β and the γ subunit. For example, γ2 dimerizes with both β1 and β2, γ1 with β1, but not with β2. To identify the structural elements of γ subunits relevant to the selectivity of βγ dimerization, we have used the baculovirus‐insect cell‐expression system to produce chimeric β and γ subunits and have studied their dimerization using an assay based on the ability of isoprenylation‐resistant γ subunit mutants to draw β subunits into the cytosol and including sucrose density gradient analysis of soluble recombinant βγ dimers. The results show that replacement of three consecutive residues of γ1, Cys36‐Cys37‐Glu38, by the corresponding residues of γ2, Ala33‐Ala34‐Ala35, suffices to render the mutant γ1 subunit capable of forming heterodimers with β2. The ability of mutant γ1, subunits to dimerize with β2 does not correlate with the probability of the mutated region to participate in coiled‐coil structures. The tripeptide region identified here as a critical determinant of the selectivity of βγ dimer formation is distinct from, but partially overlaps with, the region reported by Lee et al. [Lee, C., Murakami, T. & Simonds, W. F. (1995) J. Biol. Chem. 270, 8779–8784]. The results of this study, therefore, not only extend the region of γ1, selecting between β1 and β2 to the five‐residue sequence between Cys36 and Phe40, but also argue against the notion that the hydrophobic terminal residue of this motif represents the key determinant of selective βγ interaction.
Glycine Reductase of Clostridium Litorale
Authors: Kreimer Sylke , Andreesen Jan R.
Published: September 2004

A 2.8‐kb HindIII fragment, containing three open reading frames, has been cloned and sequenced from Clostridium litorale. The first gene grdA encoded the selenocysteine‐containing protein PA of the glycine reductase complex, a protein of 159 amino acids with a deduced molecular mass of 16.7 kDa. The second gene (grdB) encoded the 47‐kDa subunit of the substrate‐specific selenoprotein PB glycine that is composed of 437 amino acids. The third gene contained the 5′‐region of the gene for thioredoxin reductase, trxB. All gene products shared high similarity with the corresponding proteins from Eubacterium acidaminophilum. In both genes grdA and grdB, the opal termination codon (TGA) was found in‐frame, indicating the presence of selenocysteine in both polypeptides. Northern‐blot analysis showed that grdA and grdB are organized as one operon. Unlike Escherichia coli, no stable secondary structures of the corresponding mRNA were found immediately downstream of the UGA codons to direct an insertion of selenocysteine into the grdA and grdB transcripts of C. litorale. Instead, a secondary structure was identified in the 3′‐untranslated region of grdB.
A Novel Serine/Threonine‐Specific Protein Phosphotransferase Activity of Nm23/Nucleoside‐Diphosphate Kinase
Authors: Engel Matthias , Véron Michel , Theisinger Birgit , Lacombe Marie‐Lise , Seib Thomas , Dooley Steven , Welter Cornelius
Published: September 2004

Two human nm23 genes have been identified, designated nm23‐H1 and nm23‐H2, which encode the 88% identical nucleoside‐diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23‐H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA‐binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non‐denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn‐over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein‐histidine kinases is proposed, involving a high‐energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.
The Purification and Properties of Phosphonoacetate Hydrolase, a Novel Carbon‐Phosphorus Bond‐Cleavage Enzyme from Pseudomonas Fluorescens 23F
Authors: McGrath John W. , Wisdom G. Brian , McMullan Geoffrey , Larkin Michael J. , Quinn John P.
Published: September 2004

A novel, inducible, carbon‐phosphorus bond‐cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown on phosphonoacetate. The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa. Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 370C, respectively. The purified enzyme had an apparent Km of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3‐phosphonopropionate and phosphonoformate. The NH2‐terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences.
Peroxynitrite Formed by Simultaneous Nitric Oxide and Superoxide Generation Causes Cyclosporin‐A‐Sensitive Mitochondrial Calcium Efflux and Depolarisation
Authors: Packer Michael A. , Murphy Michael P.
Published: September 2004

Nitric oxide reacts rapidly with superoxide to form the potent oxidant peroxynitrite. Mitochondria in vivo produce superoxide and in pathological situations the amount of superoxide produced increases; therefore, in the presence of nitric oxide, mitochondria will be a major site of peroxynitrite formation. Oxidative stress induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation which may contribute to tissue damage in pathological situations. To determine whether peroxynitrite could induce calcium efflux and depolarisation we exposed mitochondria to both nitric oxide and superoxide simultaneously, thus subjecting the mitochondria to a continual flux of peroxynitrite similar to that found in pathological situations. Our results show that: (a) exposure of mitochondria to nitric oxide plus super‐oxide induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation; (b) neither nitric oxide nor superoxide on their own induce calcium efflux or depolarisation under these conditions; (c) calcium efflux and depolarisation occur when peroxynitrite production (from nitric oxide and superoxide) exceeds about 0.99±0.03 nmol peroxynitrite min‐1 mg mitochondrial protein‐1. This rate of production of peroxynitrite is similar in magnitude to superoxide formation by mitochondria in pathological situations, suggesting that mitochondria can produce sufficient peroxynitrite in vivo to cause mitochondrial calcium efflux and depolarisation.

Our experiments suggest a plausible model for tissue damage when mitochondrial superoxide production increases, for example in ischaemia‐reperfusion injury or following exposure to neurotoxins. In the presence of nitric oxide the mitochondrial superoxide production will lead to peroxynitrite formation which induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation. This disruption to mitochondrial function may in turn contribute to cell damage and death.
Evidence for ADP‐Ribosylation‐Factor‐Mediated Activation of Phospholipase D by m3 Muscarinic Acetylcholine Receptor
Authors: Rümenapp Ulrich , Geiszt Miklós , Wahn Friederike , Schmidt Martina , Jakobs Karl H.
Published: September 2004

Activation of phospholipase D (PLD) is a cellular response to a wide variety of extracellular ligands. However, the exact mechanisms that link cell surface receptors to PLD remain unclear. In this study, we report the involvement of the small‐molecular‐mass guanine‐nucleotide‐binding protein, ADP‐ribosylation factor (ARF), in the activation of PLD by the muscarinic acetylcholine receptor (mAChR) in human embryonic kidney cells stably expressing the human m3 subtype. PLD stimulation in permeabilized cells by guanosine 5′‐O‐[γ‐thio]triphosphate (GTP[S]) was dependent on a cytosolic factor and reconstituted by purified recombinant ARF 1. Brefeldin A, a known inhibitor of the ARF guanine‐nucleotide‐exchange‐factor activity in Golgi membranes, inhibited mAChR‐stimulated PLD, whereas basal PLD activity and stimulation by GTP[S] were not affected. Upon cell permeabilization without the addition of stimulus, ARF proteins were released. However, the addition of GTP[S] during permeabilization and mAChR activation before permeabilization caused an almost complete and partial (about 60%) inhibition, respectively, of ARF release, indicating that ARF proteins are activated and thereby translocated to membranes. The results indicate that ARF proteins and their nucleotide‐exchange factor are apparently involved in the signalling pathway leading from mAChR activation to PLD stimulation in human embryonic kidney cells.
Monoclonal Antibodies Against Two Electron Reduced Riboflavin and a Quantification of Affinity Constants for this Oxygen‐Sensitive Molecule
Authors: Bruggeman Yvonne E. , Schoenmakers Ronald G. , Schots Arjen , Pap Everard H. W. , Van Hoek Arie , Visser Antonie J. W. G. , Hilhorst Riet
Published: September 2004

In order to create a protein environment that binds preferentially to the two‐electron reduced form of flavin, monoclonal antibodies have been raised against a reduced flavin derivative. Due to the low fluorescence quantum yield and visible light absorption and to the instability of reduced flavin in an aerobic environment, it is not possible to determine the affinities of these antibodies for two‐electron‐reduced flavin using standard techniques. Because of its sensitivity, time‐resolved fluorescence can be used to overcome this problem. This technique has been applied to study the binding of two antibodies, an IgG1 and an IgM, to reduced riboflavin (1,5‐dihydroriboflavin) and oxidized riboflavin (riboflavin). The affinity of the IgG1 is more than 80 times larger for 1,5‐dihydroriboflavin than for riboflavin. From analysis of the dynamical parameters of the system it is apparent that the internal motion of 1,5‐dihydroriboflavin bound to IgG1 is much more restricted than that of riboflavin. In contrast, the affinity of the IgM is only slightly higher for 1,5‐dihydroriboflavin than for riboflavin and the flexibility of binding of both flavin redox states in the antigen binding site is almost similar.
Purification and Characterization of a Galactose‐1‐Phosphate: UDP‐Glucose Uridyltransferase from the Red Alga Galdieria Sulphuraria
Authors: Gross Wolfgang , Schnarrenberger Claus
Published: September 2004

The galactose‐1‐phosphate uridyltransferase of the red alga Galdieria sulphuraria has been purified about 1800‐fold to a final specific activity of approximately 140 U/mg protein. The purification involved chromatography on DEAE‐Fractogel, hydroxyapatite, decyl‐agarose, and DEAE‐Tentacle gel. After SDS/PAGE, the enzyme preparation showed only one protein band of 42 kDa. The enzyme is a homodimer with a molecular mass of 82 kDa as estimated from the sedimentation velocity or 60 kDa as estimated by gel filtration. It has a broad pH optimum between pH 7 and pH 9. The apparent Km values for the forward and backward reactions are Km(Glc1P) = 105 μM, Km(UDP‐galactose) = 30 μM, Km(Gal1P) = 400 μM, and Km(UDP‐Glc) = 20 μM. The activation energy of the reaction is 45 kJ mol‐1. The enzyme is specific for the galactose 1‐phosphate to UDP‐galactose interconversion in the Leloir pathway while the alternate enzyme for the Isselbacher pathway, UDP‐galactose pyrophosphorylase, could not be detected in G. sulphuraria.