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Biomedical subjects

M Paloheimo

Publications and source records attributed to M Paloheimo.

16 recordsLinked to original sources

Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis.

The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens alpha-amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.

Amino Acid Sequence

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.

Antibodies, Monoclonal

The conserved terminal region of Trichoderma reesei cellulases forms a strong antigenic epitope for polyclonal antibodies.

The specificity of polyclonal antibodies (Pab) raised against Trichoderma reesei cellulases has been studied. cDNAs lacking regions coding for certain functional domains were produced by preparing series of 3'-end deletions from the cDNAs for two cellobiohydrolases, CBH I and CBH II, and an endoglucanase, EG I. The proteins coded by the full length cDNAs and the truncated proteins coded by the deleted cDNAs were expressed in yeast Saccharomyces cerevisiae, under the control of the ADC1 promoter. Each polyclonal antiserum showed cross-reactivity with other cellulases. Pabs for CBH I and CBH II both recognized EG I. Pab for EG I strongly recognized both CBH I and CBH II. By analyzing the truncated proteins, we found that these antibodies were almost entirely directed against the conserved tail of the cellulase enzymes.

Antibodies, Fungal

Nucleotide sequence of the alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum.

The nucleotide sequence of the gene (apu) encoding the thermostable alpha-amylase-pullulanase of Clostridium thermohydrosulfuricum was determined. An open reading frame of 4425 bp was present. The deduced polypeptide (Mr 165,600), including a 31 amino acid putative signal sequence, comprised 1475 amino acids, with no cysteine residues. The structural gene was preceded by the consensus promoter sequence TTGACA TATAAT, a putative regulatory sequence and a putative ribosome-binding sequence AAAGGGGG. The codon usage resembled that of Bacillus genes. The deduced sequence of the mature apu product showed similarities to various amylolytic enzymes, especially the neopullulanase of Bacillus stearothermophilus, whereas the signal sequence showed similarity to those of the alpha-amylases of B. stearothermophilus and B. subtilis. Three regions thought to be highly conserved in the primary structure of alpha-amylases could also be distinguished in the apu product, two being partly 'duplicated' in this alpha-1,4/alpha-1,6-active enzyme.

Amino Acid Sequence

Periocular anaesthesia: technique, effectiveness and complications with special reference to postoperative ptosis.

The effectiveness of periocular anaesthesia and its complications were examined in 100 successive cataract operations. The patients were divided into 3 groups according to the duration of ocular compression with an Autopressor device after administration of periocular anaesthesia. In the control group, no compression was used (C-O, n = 36 patients). In the other two groups, compression was used for 10 (C-10, n = 32) and for 20 (C-20, n = 32) min. No differences in globe or orbicular akinesia were found between the groups. At 10 min, immobilisation of the globe in different directions was attained in 60.1-84.5% of the patients. Compression for an additional 10 min did not significantly improve the akinesia. In contrast, the hitherto undescribed loss of light perception increased with time: 15 patients at 10 min and 22 at 20 min were unable to see light. Chemosis and haematomas in the upper eyelid occurred more often in C-0 than in the other 2 groups. One day postoperatively the average palpebral aperture was smaller in C-0 than in the other two groups. The frequent postoperative ptosis (74.3% on the 1st day) decreased rapidly, but on postoperative day 7, 9 patients still had ptosis. In only one patient was ptosis still recognizable at 6 weeks postoperatively. No serious complications occurred. This study demonstrates that periocular anaesthesia with ocular compression is a suitable method for cataract surgery.

Adult

Effect of ocular compression (Autopressor) on intraocular pressure in periocular anaesthesia.

One hundred successive patients were operated on for cataract under periocular anaesthesia. The patients were divided into 3 groups to study the effect of extraocular compression on intraocular pressure. In the control group (C-O), no compression was used. In the other 2 groups, compression was applied immediately after local injection of the anaesthetic for either 10 (C-10) or 20 min (C-20). In the control group, a periocular local anaesthetic increased the IOP in 27 of 36 patients, the average increase being 3.8 mmHg (at 10 min). Postanaesthetic compression of the eye led to a decrease in intraocular pressure, which is beneficial for the operative procedure. During the first 10 min, the mean intraocular pressure decreased by 3.1 and 4.0 mmHg in groups C-10 and C-20, respectively. In the C-20 group, there was a further (1.3 mmHg) reduction in intraocular pressure between 10 and 20 min.

Anesthesia, Local

Quantitative surface electromyography (qEMG): applications in anaesthesiology and critical care.

During general anaesthesia and in lowered vigilance states such as after major trauma and during heavy sedation or analgesic medication, patients' ability to communicate with their surroundings is limited. Subjective intuitional interpretation may be the only means to ascertain a patient's emotional state, mood, and pain perception. Electromyographic detection and quantification of minimal and covert facial mimic muscle activity in anaesthesiology and critical care was an interesting concept worth further evaluation. In this study, the behaviour of quantitative surface-detected electromyographic activity (qEMG) was investigated during common anaesthetic events, post-operatively, and in volunteers as well as in experimental animals. A review of the methodology includes the necessary details for reproduction of the studies, including computerized processing of numerical data available in the commercial equipment. Results from the monitoring of 218 patients, seven volunteers and 31 rats are discussed. Conclusions are based on 32 testable null-hypotheses, the earlier documented literature and the author's own experience. The qEMG signal was derived from two electrodes placed on the frontal area and on the mastoid process behind the ipsilateral ear. After amplification, the signal was filtered to obtain a portion containing electrical activity between 60-300 Hz, which was considered to represent electromyographic activity. The signals were thereafter full-wave rectified and averaged with a 1-s time constant. The output of the processing unit consisted of a graphics display and a numeric computer output. A variety of clinical conditions and drug effects were studied in order to evaluate the method's applicability in research and in routine anaesthetic practice. The facial muscles turned out to be less sensitive to the effects of neuromuscular blocking drugs than the hand muscles, the normal monitoring site of neuromuscular transmission. Although muscle relaxants had a suppressing effect on spontaneous EMG activity, they did not abolish the ability of facial muscles to react to noxious stimuli. Also abdominal muscles retained this capability, though presumably through a different motor mechanism. Depolarizing and non-depolarizing neuromuscular blocking drugs had disparate effects on spontaneous EMG activity; succinylcholine seemed to facilitate the qEMG amplitude during recovery of the block. Quantitative surface electromyography revealed impending arousals during lightening anaesthesia, although this was not a consistent phenomenon. Inadequate anaesthesia was always reflected by an increase in facial qEMG, albeit this often was also evident to the naked eye. The arousal at the end of anaesthesia was always associated with an abrupt increase in facial qEMG activity, which often was preceded by a more gradual, predictive rise. Auditory stimulation was also effective in increasing qEMG.(ABSTRACT TRUNCATED AT 400 WORDS)

Anesthesia

Monitoring of the inspired and end-tidal oxygen, carbon dioxide, and nitrous oxide concentrations: clinical applications during anesthesia and recovery.

Respiratory oxygen, carbon dioxide, and nitrous oxide concentrations were recorded in 20 patients breath-by-breath during general anesthesia and early recovery, using the Cardiocap multiparameter monitor. Several approved maneuvers were performed to demonstrate the usefulness of endtidal oxygen measurement. "Oxygrams" provided by the fast paramagnetic oxygen sensor confirmed the capnometric information in the diagnosis of hypoventilation, apnea, and disconnections. In one patient, the alarm for inspiratory oxygen concentration, set at 18%, appeared to prevent alveolar hypoxia and low arterial saturation from occurring when oxygen instead of nitrous oxide was turned off. Low end-tidal oxygen levels revealed inadequate fresh gas oxygen supplementation while low flow circuits were closed. During manual hypoventilation at the end of anesthesia, the inspiratory-expiratory oxygen difference increased almost twofold while end-tidal carbon dioxide increased by only 30%. Changes in nitrous oxide concentration often complemented oxygen-related information obtained in our observations. In the recovery room, a decrease in end-tidal oxygen concentration preceded low pulse oximetry readings. Therefore, it is suggested that all three gases should be monitored continuously to prevent mishaps related to insufficient ventilation and inappropriate gas concentrations during anesthesia and immediate recovery.

Abdomen

Inspiratory end-tidal oxygen content difference: a sensitive indicator of hypoventilation.

The effect of progressive hypoventilation on end-tidal gas concentrations and corresponding partial pressures in arterial blood was studied in anesthetized pigs. Oxygen, CO2, and nitrous oxide concentrations were measured continuously with fast infrared and paramagnetic sensors as ventilation was decreased gradually in 12.5% increments at 5-min intervals. Samples for blood gas determinations were obtained at 3 min after each respirator adjustment. An increasing difference between inspiratory and end-tidal oxygen concentrations (FIO2 - PetO2) was the most sensitive indicator of hypoventilation and exceeded the sensitivity of end-tidal CO2. Decreasing PetO2 was followed by a decrease in PaO2, but no detectable change in arterial oxygen saturation until the ventilation was decreased to 37% of the initial value. The rapidly decreasing alveolar oxygen was replaced by nitrous oxide, and a sudden drop in ventilation was characterized by a change in the end-tidal values of all the three gases and peaked waveforms. Thus, PetO2 and FIO2 - PetO2 are sensitive and valuable indicators of adequate ventilation and appropriate oxygen supply.

Animals

Cloning and expression of the Clostridium thermohydrosulfuricum alpha-amylase-pullulanase gene in Escherichia coli.

An alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7.0 kb EcoRI fragment using a lambda vector. The gene produced, from an indigenous promoter, active thermostable alpha-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E. coli. Gel filtration separated the active enzyme produced into three peaks, each having both alpha-amylase and pullulanase activities. Immunoblotting after SDS-PAGE revealed more than ten alpha-amylase-pullulanase specific polypeptides; the biggest of these had an Mr of about 165,000, whereas the smallest enzymically active polypeptide had an Mr of about 100,000. Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80-85 degrees C) was only some 5 degrees C lower and the heat stability the same as that of the extracellular alpha-amylase-pullulanase produced by the native host. Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7.0 kb DNA insert.

Bacterial Proteins

Halothane-induced variability in the neuromuscular transmission of patients with myasthenia gravis.

The purpose of the present clinical study was to explore the skeletal muscle mechano (MMG)- and electromyographic (EMG) responses during halothane/oxygen/air anaesthesia in patients with myasthenia gravis (MG) compared with patients with normal neuromuscular transmission. The majority of MG-patients had a significant decremental response of the evoked muscle action potentials to a train-of-four (TOF) stimulation during halothane exposure (mean decrease of train-of-four ratio was 33% during the highest mean halothane concentration of 1.9 MAC). An excellent correlation was found between MMG- and EMG-measurements (r2 = 0.878, P less than 0.001). However, marked individual variations in the neuromuscular response to halothane were seen. Neither preoperative muscle fatigability nor acetylcholine receptor antibodies predicted the decremental muscle responses produced by halothane among MG-patients. The increased presence of HLA-B8 among myasthenics with halothane-suppressed muscle responses after TOF stimulation could be demonstrated (P less than 0.01).

Adolescent

Magnetically evoked facial nerve potential.

Facial nerve stimulation by electrical current is painful and tends to discourage serial studies. Transcutaneous magnetic stimulation of the facial nerve is painless, easily reproducible, and elicits facial muscle responses identical to electrical stimulation.

Electromyography

Assessment of anaesthetic adequacy with upper facial and abdominal wall EMG.

We compared changes in biopotentials arising from upper facial (FEMG) and abdominal (AEMG) muscles associated with alterations in alveolar enflurane concentration and neuromuscular block. Induction of anaesthesia significantly reduced both FEMG and AEMG mean amplitudes (-60% and -43%, respectively). Neuromuscular blocker-induced abolition of the electrically evoked thenar EMG response did not prevent FEMG and/or AEMG activation during endotracheal intubation. Decreasing the alveolar enflurane concentration was associated with an increase in FEMG amplitude prior to visible signs of arousal in half of the patients. Movement and other signs of inadequate anaesthesia were associated with distinct increases in FEMG amplitude in 29 out of 30 patients. Recovery from neuromuscular block during unchanged alveolar enflurane concentration was associated with increasing amplitudes of both FEMG and AEMG. Finally, very low-amplitude FEMG recordings were always associated with relaxed abdominal muscles.

Abdominal Muscles

Open scavenging systems.

A mathematical model was developed to cover the dynamic characteristics of open scavenging systems. The performance of such a device depends on the expiratory tidal volume (VT), the scavenging flow (Vs) and the mode of exhalational volume build-up. A widely used open scavenging system was tested by using carbon dioxide in oxygen as a marker gas. Capnography revealed gross turbulence in the expiratory reservoir, which tends to increase the polluted gas volumes and loads extra requirements onto the scavenging equipment. Increases in the scavenging flow or the expiratory reservoir volume are the aids in obtaining satisfactory safety margins for pollution-free scavenging of surplus anaesthetic gases. The theoretical basis of a new method to determine the expiratory time constant of the respiratory system is also presented.

Air Pollutants