PubMed Health⌕ Search

Biomedical subjects

F Hardy

Publications and source records attributed to F Hardy.

At least 19 recordsLinked to original sources
PubMed1997

Mutational analysis of a surface area that is critical for the thermal stability of thermolysin-like proteases.

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55-69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57-->Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.

Amino Acid Sequence↗
PubMed1997

Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay.

A reliable ELISA based method has been developed for measuring in solution antigen/antibody association rate constants. Its rationale is as follows: antigen and antibody are mixed in solution to initiate the association. At different time intervals aliquots are withdrawn to determine by an indirect ELISA the amount of free antibody that remains in solution. The disappearance of the free mAb reflects the time course of the association reaction. To test the validity of this method, the association rate constant of a monoclonal antibody for its antigen was measured and compared with that obtained previously by a method using fluorescence. The good agreement between the results obtained with the ELISA-based method and those obtained previously by fluorescence measurement indicates that the method described permits determination of true association rate constants in solution. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or the antigen, and it can be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.

Animals↗
PubMed1997

Model building of a thermolysin-like protease by mutagenesis.

The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub). An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus thermoproteolyticus (thermolysin) and Bacillus cereus as templates. The largest portion of NP-sub could be modelled satisfactorily, using standard techniques, but several surface-located regions could only be modelled with a high degree of uncertainty. In order to make the model more reliable in these regions a 'model building by mutagenesis' approach was adopted. Mutations were designed such that their effect on thermal stability could indicate how their local environment should be modelled. This approach provided insight in the local structure of several regions in NP-sub that were hard to model on the basis of homology with the two known structures alone.

Amino Acid Sequence↗
PubMed1994

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease.

Using genetic techniques the contribution of surface loops to the thermal stability of Bacillus subtilis neutral protease (NP-sub) was studied. Mutations were designed to make the surface of NP-sub more similar to the surface of more thermostable neutral proteases such as thermolysin (TLN). The mutations included the replacement of an irregular loop by a shorter variant and the introduction of a ten-residue beta-hairpin. In general, these drastic mutations had little effect on the production and activity of NP-sub, indicating the feasibility of major structural rearrangements at the surface of proteins. In the most stable mutant, exhibiting an increase in thermal stability of 1.1 degree C, approximately 10% of the surface of NP-sub was modified. Several NP-sub variants carrying multiple mutations were constructed. Non-additive effects on thermal stability were observed, which were interpreted on the basis of a model for thermal inactivation, that emphasizes the importance of local unfolding processes for thermal stability.

Amino Acid Sequence↗
PubMed1993

Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines.

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by proline. The mutations caused large positive (Ser-65-->Pro, Ala-69-->Pro) or negative (Thr-63-->Pro, Tyr-66-->Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.

Bacterial Proteins↗
PubMed1991

Cephalic and extracephalic pressure pain thresholds in chronic tension-type headache.

Pressure pain thresholds were assessed with an algometer (Somedic Inc.), over the forehead, temple and suboccipital region as well as over the Achilles tendon. A group of 32 patients suffering from chronic tension-type headache was compared to 20 healthy controls and to 10 migraineurs without aura. Although individual values were widely scattered, pressure pain thresholds were on average significantly lower in chronic tension-type headache, not only at pericranial sites but also over the Achilles tendon. Only 50% of these patients had one or more pericranial thresholds 1.5 S.D. below the mean of controls. After muscular biofeedback therapy, all pain thresholds were on average increased. Along with results obtained previously, the present data support the hypothesis that diffuse disruption of central pain-modulating systems, possibly due to a modified limbic input to the brain-stem, is pivotal in the pathophysiology of chronic tension-type headache.

Adult↗
PubMed1990

Omeprazole for prophylaxis of acid aspiration in elective surgery.

The aim of the study was to determine whether a single oral dose of omeprazole 40 mg is effective in increasing the pH of gastric residue above 2.5 at the time of anaesthetic induction in adult patients scheduled for elective gynaecological surgery. The patients were allocated to receive either chlorazepate dipotassium 25 mg alone or omeprazole 40 mg and chlorazepate dipotassium 25 mg on the night before surgery. Gastric volume and pH were measured after induction of anaesthesia. Patients who received omeprazole had a higher mean pH than control patients (p less than 0.001). The pH was less than 3.5 in 50% of patients in the control group, but in only 4.5% of those who received omeprazole (p less than 0.01). Mean (SEM) volume of gastric fluid was 15.2 (2.7) ml in the control group and 9.2 (1.8) ml in the omeprazole group, but the results were not statistically significant. A single dose of 40 mg omeprazole significantly decreased the number of patients at risk of aspiration pneumonitis.

Administration, Oral↗
PubMed1990

A pharmacokinetic study of high-dose metoclopramide suppositories.

The pharmacokinetics of high-dose rectal metoclopramide have been studied in nine patients after administration of 150-mg suppositories. The results have been compared to the pharmacokinetics of the drug in five patients who received the same dose of metoclopramide intravenously. Administration of a metoclopramide suppository achieved plasma drug concentrations that are associated with the effective treatment of cytotoxic drug-induced nausea and vomiting. In three patients who received the drug by both routes the systemic availability of the suppository appeared to be complete. High-dose metoclopramide suppositories are convenient and may be advantageous in the treatment of medical oncology out-patients.

Administration, Rectal↗
PubMed1990

[Hereditary angioneurotic edema and pregnancy].

Hereditary angioneurotic oedema (HANE) is an autosomal dominant disorder, relatively uncommon, in which patients experience recurrent bouts of oedema of the extremities, face and larynx. Attacks of HANE, sometimes fatal when involving the larynx may, among other causes, be triggered by the anesthetist's manipulation of the upper airway (tracheal intubation). HANE may affect women during their reproductive years and present serious problems in case of pregnancy. Labour can precipitate laryngeal oedema in case of general anesthesia with tracheal intubation indicated for a caesarean section or a forceps delivery, and/or perineal oedema after delivery. The authors describe a vaginal delivery in a patient with HANE that was accomplished under lumbar epidural anesthesia. No laryngeal or perineal attack was noted. This review article describes various treatments and discusses the features relevant and specific to pregnancy.

Adult↗
PubMed1989

[Hemodynamic effects of clonidine on the fetus and newborn infant].

This study on clonidine action on fetal HR and on BP and HR in newborns during the first 3 days of life was carried out with 10 mother-child couples in comparison with 10 other couples randomly chosen among our patients; in these last couples the mother has not been treated with this oral anti-hypertensive agent during the last 6 months of the pregnancy. According to the results, clonidine doesn't seem to have any negative action on the fetus and newborn.

Adult↗
PubMed1987

[Chronic refractory pain in cancer patients. Value of the spinal injection of lysine acetylsalicylate. 60 cases].

Several animal studies have demonstrated that pain is modulated by spinal mechanisms involving prostaglandins and that acetylsalicylic acid (ASA) administered intrathecally has an analgesic effect. We report our experience of this treatment in 60 patients with proven and advanced cancer. An isobaric solution of lysine acetylsalicylate was administered by lumbar puncture in doses ranging from 120 to 720 mg of ASA. The results were evaluated using the habitual criteria: scoring system, behaviour, consumption of analgesic drugs. In this trial the method proved astonishingly effective (78% of the cases). Analgesia was strong, almost immediate and without influence on motricity. No thermic or neurovegetative changes were noted. The effect of one injection lasted from 3 weeks to 1 month on average; it was reproduced and often more prolonged after a repeat injection. Pain associated with bone metastases seems to constitute the best indication, notably in breast and lung cancer and in myeloma. Visceral (pancreas) or neural pain requires higher doses to respond. Failures (22%) were due to such factors as insufficient dosage at the very beginning of our experience or severe depressive syndrome. The perineal and sphincteral pain of rectal cancer often resists treatment. This simple, inexpensive and very effective method with no other complication than a frequent tendency to fatigue should rank among other analgesic measures in cancer. The lack of respiratory depression is a major advantage over catheter spinal opiate analgesia. We consider that its main indications are pain associated with osteolytic metastases of adenocarcinomas, and myelomas. Owing to the absence of formal toxicological data, its use must be limited to cancer pain and to patients with a life expectancy of less than 2 years.

Adult↗
PubMed1987

[Changes in the pH and volume of gastric contents during pregnancy and labor].

We measured the volume and pH of gastric contents of 97 patients after induction of anaesthesia. The patients were divided into 3 groups: group I (N = 39): elective cesarean section, group II (N = 44): elective gynecological surgery, group III (N = 14): emergency cesarean section. The study was undertaken to ascertain whether pregnancy and labour would modify the volume and mean pH of gastric contents. Mean pH of gastric contents in the 3 groups were respectively 2.57; +/- 0.23; 3.21 +/- 0.29; and 2.77 +/- 0.42. There were no significant differences between the groups: Pregnancy and labour do not influence gastric pH. As far as gastric volume is concerned there is a significant difference between Group I and III (p less than 0.01) and Group II and III (p less than 0.01).

Anesthesia, General↗
PubMed1987

[Acute cerebral ischemia: an uncommon complication of ritodrine?].

Cerebral ischaemia is reported in a 33 year-old woman at 34 weeks gestation during an infusion of ritodrine to stop premature labour. CT Scan revealed multiple ischemic bilateral lesions. These lesions disappeared completely under artificial ventilation and thiopental infusion. The authors discuss the other possible causes of cerebral ischaemia in pregnant patients.

Acute Disease↗
PubMed1985

[Torsades de pointes and hypomagnesemia].

The authors report a case of "Torsades de pointe" occurring in a patient subjected to intensive surgical care and presenting several causes of magnesium loss: mutilating abdominal surgery, exteriorized gastric fistula, administration of Henle's loop diuretics, electrolytic disorders and exclusive parenteral nutrition without any magnesium Mg supplement. The arrhythmia appeared in the third week and the usual treatment was ineffective. The level of plasma magnesium was below the measurable values on three separate occasions. The administration of magnesium sulphate corrected the dysrhythmia which did not recur. Experimental and clinical data demonstrating the antiarrhythmic effect of magnesium are recalled. The electrophysiological explanation is based on the inhibition of potassium and calcium flow across the cell membrane; magnesium ion has a stabilizing membrane effect and an antagonistic effect on ectopic automaticity. They are particularly important on slowly depolarizing tissue (nodal cells) or partially depolarized cells (ischaemic or digitalis treated cardiac tissue). In intensive care, circumstances for magnesium depletion by digestive or renal losses and by lack of parenteral magnesium supplement are frequently combined. Systematic intravenous administration of magnesium is necessary when such conditions are found and whenever ventricular or auricular dysrhythmias occur.

Calcium↗