The roles of uridine-cytidine kinase and CTP synthetase in the synthesis of CTP in malignant human T-lymphocytic cells.

The pattern of incorporation of [14C]uridine showed that in MOLT-3 cells an increased proportion of CTP was synthesized via CTP synthetase, compared to proliferating normal human T lymphocytes at a physiological concentration of cytidine (< 0.5 microM). Furthermore, in the proliferating normal human T lymphocytes similar patterns of incorporation of [14C]uridine were observed in the presence of the physiological concentration of cytidine and after addition of 2 microM of cytidine. In contrast, in the MOLT-3 cells after addition of 2 microM of cytidine the proportion of CTP synthesized by conversion of UTP into CTP was substantially decreased, whereas the salvage of cytidine was proportionally increased. We conclude that the reutilization of uridine is a preferred route in the synthesis of CTP for MOLT-3 cells at physiological concentrations of uridine and cytidine, whereas in proliferating normal human T lymphocytes CTP is largely synthesized through reutilization of cytidine. This difference in salvage of pyrimidine ribonucleosides may be exploited for selective chemotherapy.

Cytidine triphosphate (CTP) synthetase activity during cell cycle progression in normal and malignant T-lymphocytic cells.

The role of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2.) in the pyrimidine ribonucleotide metabolism of MOLT-3 human T-ALL cell line cells and normal human T lymphocytes during the cell cycle traverse was studied. Highly pure G1-phase samples and samples enriched in S-phase cells were obtained by counterflow centrifugation. The activity of CTP synthetase in situ, measured in pulse-chase experiments, was similar in the G1-phase and S-phase MOLT-3 cells. In contrast, in S-phase T lymphocytes, an increased activity of CTP synthetase was observed compared with G1-phase T lymphocytes. Nevertheless, the MOLT-3 samples showed an increased activity of CTP synthetase in comparison with either G1-phase or S-phase enriched samples of normal T lymphocytes. Therefore, the increased activity of CTP synthetase of MOLT-3 cells is a cell cycle-independent feature, whereas among normal T lymphocytes, the increase in activity of CTP synthetase that arises after a growth stimulus is more prominent in the S-phase.

A fast and novel assay of CTP synthetase. Evidence for hysteretic properties of the mammalian enzyme.

A novel assay of CTP synthetase was developed which allows the processing of large numbers of samples. The amount of glutamate produced by CTP synthetase was determined with glutamate dehydrogenase and the NAD analogue acetyl-pyridine-adenine dinucleotide was used to shift the initial unfavourable equilibrium towards the formation of a-ketoglutarate. The amount of glutamate determined with the assay was comparable to that of CTP. The assay proved to be linear with time up to 90 min and protein concentrations up to 520 micrograms/ml. However, at low protein concentrations as well as at early time points a lag phase was observed. This hysteretic phenomenon of CTP synthetase may be due to the association-dissociation equilibrium that exists between the various polymerisation states of the enzyme.

Elutriation of T-lymphoblastic cells and cell cycle analysis of fractions.

Elutriation of human lymphoblastic cells has to be performed at a flow rate of 13 ml/min at a temperature of 10 degrees C to retain a high cell viability. Cell cycle analysis of elutriated cells on the basis of their DNA contents is inaccurate. The combined detection of BrdUrd incorporated into the DNA and analysis of the DNA content offers an alternative. After elutriation, fractions of cells with increased percentages of either G1 or S phase cells can be selected on the basis of the median cell size as established by a coulter counter channelyzer.

Uridine fluxes in healthy proliferating T-lymphocytes, MOLT-3 T-ALL cell-line cells and differentiated MOLT-3 cells.

Incorporation of 14C-uridine into UTP and CTP and fluxes of label through these nucleotide pools to RNA and DNA were greater in MOLT-3 cells compared to T-lymphocytes. In growth-arrested, differentiated MOLT-3 cells overall incorporation of radiolabel into nucleotides and nucleic acids was lowered compared to exponentially growing cells. Turnover of UTP and CTP however, retained the profile of exponentially growing MOLT-3 cells, implicating the characteristically higher conversion of UTP to CTP is independent of the MOLT-3 cells proliferative capacities. We conclude that drugs interfering with CTP-synthetase activity are good candidates to be used as selective substances in the battle against T-ALL.

The anesthetic technician's essential services--a training guide.

The development of sophisticated medical services in the Middle East has resulted in the need for training programmes for staff in all disciplines of health care. Operating Department Assistants (ODAs/Anesthetic Technicians) are essential operating theatre personnel, forming an integral part of the operating theatre team. This paper is designed as a teaching guide for Anesthetic Technicians in training. It lists in point form the essential services that should be provided in each of the seven identifiably different work periods in the Anesthetic Technician's working day.

Use of esmolol to attenuate hemodynamic responses during cataract extraction.

Esmolol attenuates hemodynamic responses to tracheal intubation and extubation in young patients, but has less well documented pharmacokinetics and efficacy in older patients. Following cataract surgery, application of pressure on the eye during eye bandaging may have vasomotor effects. The present study of older patients having cataract surgery investigated 1) the effects of normal saline 1.0 ml. 10kg(-1) or esmolol 4.0 mg.kg(-1) IV given 90 secs prior to tracheal intubation and of normal saline 0.5 ml.10 kg(-1) or esmolol 2.0 mg.kg(-1) IV given 60 sec prior to each of eye bandaging and tracheal extubation; 2) the time to onset and duration of action of esmolol; 3) the cardiovascular effects of eye bandaging. Esmolol attenuated the cardiovascular effects of tracheal intubation, eye bandaging and tracheal extubation, but caused relative bradycardia and hypotension after induction and hypotension after extubation. Its effect occurred within 60-90 secs and lasted about 6 mins. Pressure on the eye during bandaging in those not given esmolol caused hypertension without tachycardia.

Effects of heart rate, blood pressure and bronchomotor tone of halothane, enflurane and isoflurane in young fit patients.

A prospective study was undertaken on 169 healthy young patients undergoing elective ear, nose and throat surgery to assess the intraoperative effects on heart rate, blood pressure and bronchomotor tone of MAC in nitrous oxide concentrations of halothane (0.4 to 0.6%), isoflurane (0.6 to 0.8%) and enflurane (0.8 to 1%). All the agents produced a progressive slowing of the heart rate during anesthesia. Halothane did so most profoundly, resulting in a maximum slowing of 40 beats-1 compared with a maximum slowing of about 20 beats min-1 with both isoflurane and enflurane. The three volatile agents also produced progressively similar degrees of hypotension during anesthesia. The hypotension produced by enflurane occurred earliest and was most profound; that produced by isoflurane occurred less early and was less severe, but occurred earlier and was more severe than that produced by halothane. Neither halothane, isoflurane nor enflurane appeared to have any effect on bronchomotor tone.

Electroconvulsive therapy and intraocular pressure.

Psychiatric patients receiving phenothiazine, tricyclic antidepressant and antiparkinsonian drugs for prolonged periods, occasionally develop mydriasis and angle closure glaucoma. Suxamethonium, usually given to modify the convulsion of electroconvulsive therapy (ECT) increases intraocular pressure (IOP) by about 7-8 mmHg, the increase being maximal and having returned to baseline 2 min and 6 mins after injection, respectively. We studied the effects on IOP of an electrically induced convulsion following induction of anesthesia using methohexitone 1 mg.kg(-1) and suxamethonium 0.5 mg.kg(-1) in 21 consecutive cooperative psychiatric patients, all receiving antipsychotropic drugs. IOP was recorded sequentially from before induction of anesthesia to after resumption of spontaneous respiration. Their mean IOP was 15.3 (SD 3.7) mmHg prior to induction of anesthesia, 13.5 (SD 3.5) mmHg after loss of eyelash reflex following injection of methohexitone, 16.1 (SD 2.4) mmHg after cessation of muscle fasciculations induced by suxamethonium, 19.2 (SD 5.6) mmHg after cessation of convulsion and 15.5 (SD 4.4) mmHg following resumption of regular spontaneous respiration. The successive stepwise changes in the mean IOP were all statistically significant (p < 0.001 each change compared with the preceding pressure; paired 't' tests). These data reveal that the reduction in IOP produced by methohexitone is reversed by the increase in IOP produced by suxamethonium. Collated with the time course of the effects of barbiturates and suxamethomium on IOP, the increase in IOP observed following the induced convulsion was not greater than that expected after suxamethonium alone, suggesting that the induced convulsion during ECT does not pose an ocular hazard to psychiatric patients receiving medications which have iatrogenic glaucomatous potential.

Reducing cardiovascular responses to laryngoscopy and tracheal intubation: a comparison of equipotent doses of tramadol, nalbuphine and pethidine, with placebo.

The stress response to tracheal intubation may be obtunded by opioids given with induction of anesthesia. Tramadol is an opioid acting on mu-receptors and the monoaminergic pain modulating systems. This study examined vasomotor responses to tracheal intubation after equipotent doses of tramadol, nalbuphine and pethidine (3.0, 0.3 mg/kg(-1), and 1.5 mg/kg(-1), respectively), and placebo, given prior to induction of anesthesia in 118 healthy patients. Premedication and induction of anesthesia were standardized. Recordings of HR and SAP were made prior and subsequent to induction of anesthesia, and at 1, 3, 5 and 7 minutes after tracheal intubation. Prior to laryngoscopy and intubation, HR increased in all groups (p < or = 01, all comparisons), but least so after nalbuphine, whilst SAP remained unchanged after placebo, tramadol and pethidine, but fell after nalbuphine (p < 0.025). Maximum increases in HR (p < or = 0.005, all comparisons) and SAP (p < or = 0.02, all comparisons) occurred one minute after intubation. Maximum HR after placebo (108 SD 15 bpm), tramadol (107 SD 20 bpm), pethidine (113 SD 16 bpm) and nalbuphine (110 SD 26 bpm) was similar; with placebo HR remained faster than baseline until the seventh minute but had returned to baseline by the fifth minute with the opioids. Maximum SAP with tramadol (151 SD 26 mmHg) was similar to that with placebo (157 SD 20 mmHg), but was greater than after pethidine (136 SD 27 mmHg; p < 0.05) and nalbuphine (135 SD 19 mmHg; p < 0.02). With each test drug SAP returned to baseline by the third minute. It is concluded that, in these doses, 1) tramadol does not attenuate the chronotropic nor the inotropic response to tracheal intubation, and 2) pethidine and nalbuphine reduce only the inotropic response to airway instrumentation.

Responses to dural puncture during institution of combined spinal-epidural analgesia: a comparison of 27 gauge pencil-point and 27 gauge cutting-edge needles.

Pencil-point spinal needles are popular for combined spinal-epidural analgesia because they cause less dural puncture headache than cutting-edge spinal needles. However many parturients move, grimace, vocalise or experience paraesthesia or dysaesthesia during dural puncture when performing 'needle through needle' combined spinal-epidural analgesia. We compared dural puncture responses induced by pencil-point and cutting-edge needles (both 27 gauge). With institutional approval, 115 parturients presenting for elective caesarean section or labour analgesia were audited. After lignocaine infiltration, a Tuohy-type needle was inserted to loss of resistance to saline at a mid-lumbar interspace, and either a 27 gauge cutting-edge or 27 gauge pencil-point needle was inserted 'needle through needle' through the dura. During dural penetration, the occurrence of patient movement, grimacing or vocalisation was noted by a blinded observer, as was the patient's response to the question "Did you feel that?" asked by the anaesthetist. The audit comprised two similar groups of patients (caesarean section, n=30; labour analgesia, n=85). In both groups, grimacing and movement during thecal penetration occurred more frequently with pencil-point needles (P < 0.05 and P < 0.025, respectively). Pooled data analysis revealed that pencil-point and cutting-edge needles induced grimacing and movement in 17 (22%) and 2 (5%), spontaneous vocalisation in 4 (5%) and 1 (3%) and was perceived by 13 (17%) and 3 (8%) parturients (P < 0.025, P=NS, P=NS), respectively. Overall, 34 and 6 objective and subjective patient responses (P < 0.005) occurred when inserting these needles, respectively. Dural puncture by a 27 gauge pencil-point needle inserted 'needle through needle' when instituting combined spinal-epidural analgesia induces more iatrogenic responses than a 27 gauge cutting-edge needle.

Loss of resistance to saline reduces responses accompanying spinal needle insertion during institution of 'needle-through-needle' combined spinal-epidural analgesia.

Normal saline or air is used to identify loss of resistance during identification of the epidural space for combined spinal-epidural analgesia. Following epidural needle placement using air for loss of resistance, up to 80% of parturients move, grimace, vocalise or experience paraesthesia or dysaesthesia during subsequent dural puncture by a spinal needle. We compared the effects of saline versus air for loss of resistance on the occurrence of these subjective and objective responses during thecal penetration. With institutional approval, 55 parturients presenting for labour analgesia were studied. After infiltration of lignocaine at an L2-L5 vertebral interspace, a 17 gauge Tuohy epidural needle attached to a 5 ml loss of resistance syringe containing either saline or air was inserted and advanced until loss of resistance was identified by injection of 3 to 5 ml of content. During subsequent 'needle-through-needle' insertion of a 27 gauge pencil-point spinal needle through the meninges, all subjective and objective patient responses were recorded, as well as each patient's reply to the question "Did you feel that?". The two groups (n = 28, n = 27) were comparable. In those given saline and air respectively, 5 (18%) and 12 (44%) parturients responded to and/or acknowledged having perceived dural puncture (P < 0.005). Overall, 7 and 31 (P < 0.0005) subjective and objective responses occurred during dural puncture in those given saline and air, respectively. The study found that use of saline to determine loss of resistance is associated with fewer patient responses at the moment of thecal penetration during 'needle-through-needle' placement of the spinal needle at combined spinal-epidural analgesia.

Bradycardia and hypertension in anticipation of, and exacerbated by, peribulbar block: a prospective audit.

BACKGROUND: Changes in heart rate (HR), systolic arterial pressure (SAP) and diastolic arterial pressure (DAP) occur in anticipation of, and following, injection of a peribulbar local analgesic agent. We examined these changes in two groups of awake patients given a pre-medication of either hydroxyzine 1.0 mg/kg alone (control) or hydroxyzine 1.0 mg/kg with morphine 0.05 mg/kg. METHODS: HR, SAP and DAP of 100 patients per group were monitored the day before surgery (baseline), every 5 min in the anesthesia holding room before peribulbar injection, every minute for the first 5 min after peribulbar injection and then every 5 min until transfer to the operating room. Within and between pre-medication group values of HR, SAP and DAP before and after peribulbar injection were compared with baseline. RESULTS: The two groups of patients were similar. Before peribulbar injection, HR was unchanged in the hydroxyzine group, but 6% slower in those given morphine (P<0.01). After injection, HR slowed in both groups, by 5% and 7% (P<0.01, both comparisons), respectively. In anticipation of injection, SAP increased in both groups to 20% and 16% above baseline, respectively, and increased further after injection to 26% and 24% above baseline, respectively (P<0.001, all comparisons). In both groups, maximum SAP following injection exceeded maximum SAP before injection (P<0.02, both comparisons). DAP increased by 4% (P<0.05) in the hydroxyzine group before injection, and by 5% and 4%, respectively (P<0.005 and P<0.05, respectively) after peribulbar injection. CONCLUSION: The audit reveals pronounced increases in SAP accompanied by lesser increases in DAP and a tendency to slowing of HR in awake patients in anticipation of peribulbar injections. Peribulbar injections cause further increases in blood pressure and mild bradycardia. These changes occur similarly in patients pre-medicated with hydroxyzine or hydroxyzine plus morphine. A mix of neuro-humoral influences (anxiety/catecholamine/baroreceptor/trigemino-vagal) are postulated as etiological.

A comparison of two double-injection techniques for peribulbar block analgesia: infero-temporal plus supero-medial vs. infero-temporal plus medial-percaruncular.

BACKGROUND: Combinations of infero-temporal and either supero-nasal ('inferior-superior') or medial percaruncular ('inferior-medial') injections are popular double-injection techniques for establishing peribulbar block analgesia. This study compared the efficacy of these two techniques in achieving ocular and lid akinesia. METHODS: One hundred patients were randomized to receive inferior-superior or inferior-medial injections in a study in which injectate, injectate volumes, 5-min ocular akinesia scoring (0-8), lid scoring (0-2) and supplemental injection protocols were standardized. The numbers of supplemental injections required at each observation period and the total volume of injectate required to produce ocular and lid akinesia were compared. RESULTS: The two test groups were demographically similar. The inferior-medial combination achieved greater ocular akinesia than the inferior-superior combination 5 min after the initial injections (mean score +/- standard deviation of 1.74 +/- 1.86 vs. 2.66 +/- 2.39; P < 0.05), with a reduced requirement for supplementary injections (3 vs. 23 supplementary injections; P < 0.025). The inferior-superior technique achieved greater lid akinesia at 5 min than the inferior-medial technique (mean score +/- standard deviation of 0.7 +/- 0.9 vs. 0.3 +/- 0.58; P < 0.005). A medial subconjunctival hemorrhage occurred in one patient in the inferior-medial group. CONCLUSION: Compared with the inferior-superior technique, the inferior-medial combination achieved more rapid ocular akinesia with less need for supplementation, but induced less efficient lid akinesia and had a propensity to cause iatrogenic subconjunctival hemorrhage. The latter complication is considered by our surgeons to be a contraindication to the inferior-medial technique in patients undergoing trabeculectomy.