Biomedical subjects

M Laub

Publications and source records attributed to M Laub.

At least 19 recordsLinked to original sources

PubMed1999

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Culture Media↗

PubMed1999

Anaesthetic implications of rigid spine syndrome.

The perioperative management of a 14-year-old girl, suffering from the muscular disorder rigid spine syndrome, is presented. The anaesthetic implications with regard to possible difficult intubation, cardiac involvement, malignant hyperthermia, neuromuscular blocking agents, and postoperative recovery are discussed.

Adolescent↗

PubMed1998

Modulation of calmodulin function by ubiquitin-calmodulin ligase and identification of the responsible ubiquitylation site in vertebrate calmodulin.

Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell-free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl-calmodulin synthetase (ubiquitin-calmodulin ligase, EC 6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+-binding loop of calmodulin and has the octapeptide structure -L-F-D-K21-D-G-D-G- with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl-calmodulin synthetase. Removal of the 41 C-terminal amino acids (fourth Ca2+-binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site-specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site -V-F-D-K94-D-G-N-G- in the third Ca2+-binding loop is apparently not ubiquitylated by the synthetase.

Amino Acid Sequence↗

PubMed1998

The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the ubiquitin-binding first component, a ubiquitin-activating enzyme.

Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.

Amino Acid Sequence↗

PubMed1998

The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the calmodulin-binding second component and enzymatic properties.

Ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+-dependent reversible synthesis of ubiquityl-calmodulin which is not directed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components: uCaM Syn-F1, a ubiquitin-binding protein belonging to the ubiquitin-activating enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn-F2, which binds to calmodulin-Sepharose in a Ca2+-dependent manner, has been purified over 3500-fold in seven steps from rabbit reticulocytes and has a native molecular mass of approximately 620 kDa. It binds calmodulin with a Km of 5 microM and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn-F2 is 6-8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I-ubiquitin and ATP/Mg2+ were determined to be 8 microM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin-Sepharose, two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two-component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.

Animals↗

PubMed1998

Serum concentrations of lignocaine and its metabolite monoethylglycinexylidide during fibre-optic bronchoscopy in local anaesthesia.

Fibre-optic bronchoscopy was performed in local anaesthesia using lignocaine. Serum concentrations of lignocaine and its active metabolite monoethylglycinexylidide (MEGX) were measured in 16 patients at regular intervals up to 120 min after administration. Lignocaine was administered as an aerosol in the upper respiratory tract and as a solution in the bronchial tree. The total dose of lignocaine ranged from 243 to 608 mg (2.4-8.0 mg kg-1 body weight). The dose of lignocaine given as an aerosol ranged from 163 to 508 mg (1.6-6.6 mg kg-1) and the dose given as a solution ranged from 60 to 180 mg (0.8-2.5 mg kg-1). The highest median serum lignocaine concentration, 10.5 mumol l-1, was measured 20 min after administration. None of the patients had toxic serum lignocaine levels (> 26 mumol l-1) or adverse effects. The highest median serum MEGX concentration, 1.7 mumol l-1, was measured 120 min after administration. The dose of lignocaine, expressed in mg per kg body weight correlated with serum lignocaine and serum MEGX (rs = 0.47 and rs = 0.39, respectively). Lignocaine is a clinically safe, local anaesthetic agent provided the total dose does not exceed 6-7 mg kg-1 body weight.

Aged↗

PubMed1997

Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues.

Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40,000 x g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4-80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3-4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse of 30 min was followed in liver crude extracts by the addition of EGTA, which specifically inhibits ubiquityl-calmodulin synthesis, a half-life of calmodulin-conjugate decay of 15-20 min is observed. A similar conjugate half-life of ca. 30 min was observed after addition of EDTA excluding that conjugate decay is due to an ATP-dependent proteolytic process. Studying the decay of purified ubiquitin-125I-BH-calmodulin conjugates in cell-free reticulocyte extracts led to the discovery of an ATP-independent isopeptidase activity which splits ubiquitin-calmodulin conjugates without leading to detectable calmodulin fragments. The rapid decay of ubiquitin-calmodulin conjugates in tissue extracts can therefore be plausibly explained by a ubiquityl-calmodulin splitting isopeptidase activity.

Adenosine Triphosphate↗

PubMed1997

Treatment of ventricular fibrillation during anaesthesia in an anaesthesia simulator.

BACKGROUND: To evaluate treatment of ventricular fibrillation (VF) occurring during anaesthesia and the use of a full-scale simulator, 80 anaesthetists in teams of two were attending a training session in the simulator Sophus. METHODS: The sessions were recorded on videotape and reviewed with the anaesthetists afterwards. Time of treatment and the sequence were registered. RESULTS: Onset time for VF was the starting point. Most of the subjects changed respiratory settings. Four teams did not change inspiratory oxygen and 17 teams did not turn off the vaporiser. Cardiac compression was initiated by all teams. DC-defibrillation was not used by two teams, with 38 of 40 teams defibrillating once, 37 twice and 29 teams three times. Adrenaline was administered by 30 of 40 teams. CONCLUSION: There was very little consistency among the teams regarding treatment for VF according to accepted algorithms. An anaesthesia simulator could be a tool for training and it is a safe way of demonstrating for the anaesthetist that certain treatment algorithms and behaviour during critical incidents are the most effective.

Anesthesia↗

PubMed1993

[Autoimmune cytopenia in pernicious anemia].

Authors report on their 5 patients with pernicious anaemia whose autoimmunocytopenia was diagnosed at different times after the diagnosis and treatment of their original disease. All of the patients were women. They have found autoimmune thrombocytopenic purpura in two cases, agranulocytosis in two cases and autoimmune haemolytic anaemia in one case. They compare their patients with the cases of literature.

Adult↗

PubMed1993

A ubiquityl-calmodulin synthetase that effectively recognizes the Ca(2+)-free form of calmodulin.

Ubiquityl-calmodulin synthetase (uCaM-synthetase) activity as detected in reticulocyte lysate and the crude extracts of rabbit tissues [FEBS Lett. 294 (1991) 229-233] has been well characterized as being essentially Ca(2+)-dependent (-Ca2+/+Ca2+ activity ratio: 0.15-0.2). However, during the purification of this enzyme on ubiquitin-Sepharose the Ca(2+)-dependent activity is lost and an essentially Ca(2+)-independent enzyme (-Ca2+/+Ca2+ activity ratio: 1.0-1.5) is obtained which was purified 90-fold (uCaM-Syn F1) to a final specific activity of 0.32 pkat/mg. During the purification procedure a second protein factor (uCaM-Syn F2) was isolated that has no catalytic activity by itself but restores Ca2+ dependence to the uCaM-Syn F1 fraction (-Ca2+/+Ca2+ activity ratio: 0.1) and enhances the catalytic activity in uCaM-Syn F1 in the presence of Ca2+ over 40-fold. It is concluded that several (possibly interdependent) forms of uCaM-synthetase exist which display different substrate specificities for calmodulin.

Animals↗

PubMed1993

Molecular characterization of beta-thalassemia in Hungary.

We have identified seven different beta-thalassemia mutations and one delta beta-thalassemia determinant (the Sicilian type) in 32 members of 17 Hungarian families. The most common mutation is the IVS-I-1 (G-->A) change; its high frequency is comparable to that observed in neighboring Czechoslovakia. Additional mutations are of Mediterranean origin. One rare mutation (initiation codon ATG-->GTG) was identified as an independent mutation because of the absence of known polymorphisms in the beta-globin gene. One new frameshift at codon 51 (-C) was observed in a single individual; hematological data were as expected for a beta zero-thalassemia heterozygosity.

Amino Acid Sequence↗

PubMed1993

Three different mask physiotherapy regimens for prevention of post-operative pulmonary complications after heart and pulmonary surgery.

OBJECTIVE: An investigation into the incidence of post-operative complications after thoracic surgery with 3 different physiotherapy masks. DESIGN: A prospective, consecutive, randomized comparison. SETTING: Department of Thoracic and Heart Surgery at a University Hospital. The treatments were performed by experienced and specially trained physiotherapists. PATIENTS: 160 patients were evaluated. 60 patients undergoing heart surgery, 59 patients having pulmonary resection, and 41 patients with exploratory thoracotomy. INTERVENTIONS: In each operative category the patients were treated with one of three face mask systems used in addition to routine chest physiotherapy. These were either continuous positive airway pressure (CPAP), positive expiratory pressure (PEP), or inspiratory resistance - positive expiratory pressure (IR-PEP). MEASUREMENTS AND RESULTS: Post-operative pulmonary complications were assessed by forced vital capacity (FVC), arterial oxygen tension (PaO2), and chest X-ray examination, all measured pre-operatively and on the fourth and ninth post-operative day. The patients filled in a questionnaire expressing their opinion about their mask treatment. There was an equal decrease in FVC, FVC%, and PaO2, and equal frequency of atelectasis in the 3 mask treatments. More patients with the PEP mask favoured their system than did those with the other 2 systems. CONCLUSION: There was no statistically significant difference between the treatments: continuous positive airway pressure (CPAP), positive expiratory pressure (PEP), and inspiratory resistance - positive expiratory pressure (IR-PEP) on post-operative complications. Any of the three treatments may be used as supplement to standard chest physiotherapy.

Blood Gas Analysis↗

PubMed1993

Spleen emptying and venous hematocrit in humans during exercise.

The spleen may release pooled erythrocytes to the general circulation during strenuous conditions such as heavy exercise. Most of our knowledge of this reservoir function of the spleen derives from animal studies, and the splenic contribution to the circulating blood volume in humans has been regarded as unimportant. We recorded the erythrocyte content in the human spleen during graded bicycle exercise to maximal working capacity. In five normal adults 99mTc-labeled autologous erythrocytes were injected intravenously, and the subjects were placed on bicycles with the back against a gamma camera focusing on the spleen. During increasing exercise the splenic erythrocyte content decreased linearly, and at maximal work load it had been reduced to a mean of 34.2% (range 44-26%) of the initial count rate at supine rest. Concomitantly norepinephrine and epinephrine in plasma increased gradually, whereas neuropeptide Y increased only at maximal exercise. A rise in hematocrit from a mean of 44.6 to 48 was observed, but the autotransfusion of erythrocytes from the spleen only partly explains the rise in hematocrit during physical activity.

Adult↗

PubMed1992

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.

Animals↗

PubMed1992

Sublingual premedication with brotizolam.

This randomized, double-blind and double-dummy study was carried out in order to compare the perioperative sedation after premedication with either brotizolam 0.25-0.50 mg sublingually or diazepam 5-10 mg orally. Sixty-two patients aged 18-60 years scheduled for minor gynaecological surgery in general anaesthesia were included. Assessments were: 1. auditory continued response time (ACRT); 2. coma scale; 3. anxiety scale; and 4. final patient questionnaire. One hour after premedication the brotizolam group was more sedated, based on ACRT (P < 0.01) and the coma scale (P < 0.05). The final questionnaire showed (P < 0.05) that the brotizolam group was more satisfied with the effect of the premedication. Seven hours after the premedication the ACRT scores in both groups were similar to those before premedication and all the patients could walk about freely. In conclusion, as a premedicant in outpatients sublingual brotizolam appears to be a good alternative to diazepam.

Administration, Oral↗

PubMed1991

Ubiquitination of endogenous calmodulin in rabbit tissue extracts.

Previously we were able to show that purified calmodulins from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner. It was therefore pertinent to answer the question if a tissue extract contains all the components necessary for the endogenous synthesis of ubiquityl calmodulin (uCaM). Therefore [125I]ubiquitin, ATP/Mg2+ and Ca2+ were added to tissue extracts enriched by a single ion exchange step. In such extracts of red blood cells, skeletal muscle and testis a novel ubiquitin conjugate of 27-29 kDa is formed. This novel band could be identified as ubiquityl-calmodulin by the following methods: (i) identical Rf-value of novel conjugate and standard uCaM in SDS-PAGE; (ii) Ca(2+)-dependent conjugate formation; (iii) Ca(2+)-dependent adsorption to fluphenazine-Sepharose; (iv) Ca(2+)-dependent mobility change of the novel conjugate during SDS-PAGE; and (v) inhibition of conjugate band formation by phosphorylase kinase. These experiments clearly demonstrate that ubiquityl calmodulin can be endogenously generated in enriched cellular extracts and strongly indicate that this reaction is of importance in vivo.

Adenosine Triphosphate↗

PubMed1990

Lytic cocktail in children. Rectal versus intramuscular administration.

The efficacy of the lytic cocktail (1 ml contains pethidine 28 mg, promethazine 7 mg, chlorpromazine 7 mg) administered intramuscularly or rectally as premedication was studied in 51 children aged 1-12 years who had minor elective otological surgery. One group received 0.05 ml/kg intramuscularly (maximum dose 2.0 ml) and the other 0.07 ml/kg per rectum (maximum dose 2.8 ml). Most were satisfactorily sedated before operation, but after operation the rectally premedicated children were less sedated, which was in agreement with lower plasma pethidine concentrations in this group. The rectal dose should be increased if prolonged postoperative sedation is desireable.

Administration, Rectal↗

PubMed1990

Role of small calibre chest tube drainage for iatrogenic pneumothorax.

A 2 mm Teflon catheter was used as a chest tube in 28 patients with iatrogenic pneumothorax. Frequent aspirations through the catheter were performed in 16 of the patients. In the remaining 12 patients the catheter was connected to a one way flutter valve. The treatment was successful in 27 of the 28 patients--one patient required a large calibre chest tube. The mean drainage time was 48 hours. The small catheter technique is superior to the use of a large intercostal drain as it is much less traumatic and troublesome. The small calibre chest tube with a one way valve is recommended as a safe and easy technique.

Adult↗