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PubMed1983

Effect of cooling rate on human and murine hemopoietic precursor cell recovery.

The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me2SO, a cooling rate of 7 degrees C/min from -4 to -30 degrees C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 degrees C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoietic precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 degrees C/min.

Animals↗

PubMed1987

Periodicity in the Hoffmann reflex recovery curve.

Hoffmann (H) reflex recovery curves were recorded from the soleus muscles of 10 healthy adult subjects with a view to further elucidating the pattern of facilitation of the motoneuron pool. Specific consideration was given to the possible existence of periodicity in facilitation of the motoneuron pool following a subthreshold conditioning H-reflex stimulus. The reliability of the recovery curves was also examined. The recovery curves, which utilized a 50% maximal test response, revealed the well established early facilitation (peak at 10 ms) followed by a later facilitation commencing 50 to 70 ms after the conditioning stimulus. In addition, there was evidence of peaks in excitability of the motoneuron pool occurring at 70 to 75 ms, 125 to 150 ms, and about 250 ms. The reliability of the periodicity in the recovery curve was established by examining the subjects on two separate occasions and investigating the effect of the number of measurements made within each day. Although there was considerable inter- and intrasubject variability in the form of the recovery curves, both within days, and between days, the overall group data showed a remarkably consistent periodicity for the first 350 ms following the conditioning stimulus. Methodological considerations that are important for observing the periodicity in the recovery curve were identified. These included an adequate number of measurements (test reflexes) per data point in the recovery curve, adequate sampling rate, and conditioning and test stimulus intensity. The clinical significance of these results resides in the possible contributions of the peripherally triggered periodic facilitation to rhythmic phenomena such as clonus and physiologic tremor.

Adult↗

PubMed1991

Do propriospinal projections contribute to hindlimb recovery when all long tracts are cut in neonatal or weanling rats?

Lateral hemisection lesions separated by 1 to 3 spinal segments were made on opposite sides of the mithoracic spinal cord in 1-month-old (N = 15; weanling operates) and newborn albino rats (N = 16; neonatal operates). Hindlimb behavior was assessed between 1 and 6 months p.o. for both groups of operates using a protocol and rating system that have previously proved effective in differentiating behavioral recovery of the hindlimbs as a function of age of spinal transection. In addition, at the conclusion of behavioral testing, operates received spinal injections of [3H]proline and HRP caudal to the spinal lesions to determine if lesions were complete and if neurons within the region between the two lesions (interlesion zone) projected into the caudal spinal cord. In both groups of operates, neurons were retrogradely labeled within the interlesion zone bilaterally, primarily in laminae VII-VIII. When both lesions were complete lateral hemisections in weanling operates, little behavioral recovery was observed, similar to complete spinal cord transection (N = 3). However, much greater behavioral recovery was seen, including supporting reactions and locomotor responses, when one or both lesions spared axons along the ventrolateral rim of the white matter. Neurons were retrogradely labeled in the brain stem reticular formation (N = 12) in these cases. All lesions were complete lateral hemisections in neonatal operates but much greater behavioral recovery was seen than in weanling operates with the same lesions, including supporting, placing, and locomotor responses. In an additional group of eight neonatal operates, the spinal cord rostral to the spinal hemisections was transected at 1 month of age. Supportive, placing, and locomotor responses were seen immediately after recovery from anesthesia and responses returned to pretransection levels in six of eight operates over the 10-day survival period. Fink-Heimer impregnation showed that degeneration argyrophilia from the transection bilaterally filled the interlesion zone but little argyrophilia was seen caudal to this region. Our results indicate that an intact propriospinal circuit remains in both neonatal and weanling operates but does not appear to contribute to hindlimb response development or recovery. The greater behavioral recovery in neonatal operates appears due to intrinsic connections (doral root, interneuronal) continuing to be able to drive the spinal circuitry underlying the spared behaviors.

Aging↗

PubMed1990

Recovery of the slow inward current from Ca2(+)-mediated and voltage-dependent inactivation in the rabbit sinoatrial node.

The process of recovery from inactivation of the slow inward current (isi) in the rabbit sinoatrial (S-A) node was evaluated by voltage clamp experiments using double microelectrode method. Double pulse experiments were carried out on preparations loaded with Cs+ using nystatin. The rate of the rapid isi inactivation depended on the amplitude of isi rather than the membrane potential. After a 50 ms depolarizing prepulse isi rapidly recovered from inactivation at -40 mV with a time constant of 84 +/- 25 ms, whereas after a longer prepulse, an additional slow recovery phase with a time constant of 1 to 2 s was observed. The amount of isi during the fast recovery phase following a 300 ms prepulse to -20 mV was smaller than that following a prepulse to +20 mV, as predicted by a Ca2(+)-mediated inactivation mechanism. Conversely, a prepulse to +20 mV caused a greater inactivation in the slow recovery phase, suggesting a voltage-dependent mechanism. The inactivation curve obtained during the fast recovery phase using 50 ms prepulses was U-shaped, indicating a predominant role of the Ca2(+)-mediated mechanism, whereas that obtained during the slow recovery phase resembled previously reported voltage-dependent steady state inactivation (f infinity) curve. These results indicate that the fast and slow phases of recovery represent the removal of Ca2(+)-mediated and voltage-dependent isi inactivation, respectively. The presence of Ca2(+)-mediated inactivation via steady state Ca2+ inflows was also suggested.

Animals↗

PubMed1991

Drug distribution studies with microdialysis: I. Tissue dependent difference in recovery between caffeine and theophylline.

Microdialysis was applied to estimate concentrations of caffeine and theophylline in vitro or in vivo in blood, adipose tissue, muscle, liver and brain of rats. The in vivo and in vitro recovery of a compound was estimated by perfusing the dialysis probe with varying concentrations of caffeine and theophylline. The difference between the concentration in the dialysate and the concentration in the perfusion medium was plotted against the concentration in the perfusion medium and the slope of the resulting line was taken as an estimate of the recovery (difference method). In all experiments caffeine (20 mg/kg sc) and theophylline (20 mg/kg sc) were administered simultaneously. The recovery in vitro was virtually identical for caffeine and theophylline. The in vivo recovery of theophylline was significantly smaller than the recovery of caffeine in brain, liver, muscle and adipose tissue. The difference in recovery was significantly larger in the brain than in other tissues. The results show that the transport of a substance from the tissue to the dialysis probe may differ between tissues and between chemically very similar compounds. It is shown that the recovery of theophylline rapidly declines after death ensues which shows that energy-dependent processes are involved in the transport to the dialysis probe and not solely passive diffusion. It is suggested the differences in transport over brain capillaries explain the difference between caffeine and theophylline. It is concluded that the use of internal standards in microdialysis experiments requires validation in every specific application.

Adipose Tissue↗

PubMedDate not supplied

Recovery of growth-arrested human fibroblasts from UV-induced lethal damage is inhibited by low cell density or sodium butyrate.

The influence of growth arrest on recovery from UV-induced lethal damage in human fibroblasts was investigated. Cells were arrested in G1 either by confluent holding (high cell density at 37 degrees C) or by temperature holding (low cell density at 30 degrees C) or by a combination of both methods. These experiments showed that survival recovery only occurred in cells arrested at high cell density, both at 37 degrees C and at 30 degrees C, and not in cells arrested at low cell density at 30 degrees C. Furthermore, it was demonstrated that the rate of transcription in unirradiated cells arrested at high density, both at 37 degrees C and at 30 degrees C, was much lower compared to that in cells arrested at low density. These observations can be explained by assuming that impaired transcription is responsible for UV-induced cytotoxicity and that recovery of RNA synthesis due to repair of damage in transcriptionally active genes may account for survival recovery. Sodium butyrate did not influence the absence of recovery during low temperature holding at low density but inhibited survival recovery during confluent holding. Sodium butyrate might prevent survival recovery at high cell density by inducing a chromatin structure resembling that of untreated cells arrested at low density.

Butyrates↗

PubMed1994

Effect of propentofylline (HWA 285) on metabolic and functional recovery in the spinal cord after ischemia.

The effect of propentofylline on metabolic and functional recovery in the spinal cord after ischemia and reperfusion was investigated. Ischemia was induced by abdominal aorta ligation below the left renal artery for 20 or 30 min. Propentofylline (1, 5, 10 and 20 mg/kg) was administered intravenously, immediately after reperfusion and the animals recovered for 4 days. Propentofylline at a dose of 1 mg/kg and 5 mg/kg had only a slight effect on energy metabolism recovery in the spinal cord and neurological recovery of hindlimbs. However, almost complete recovery of adenine nucleotides, lactate and glucose occurred after 20 min of ischemia in the animals treated with 10 or 20 mg/kg propentofylline. Partial metabolic recovery occurred even after 30 min of ischemia and 20 mg/kg propentofylline. The recovery of energy metabolism correlated closely with the recovery of neurological functions after ischemia and 4 days of survival.

Analysis of Variance↗

PubMed1987

Recovery of porcine aortic endothelial cell prostaglandin synthesis following inhibition by sublethal concentrations of hydrogen peroxide.

Confluent monolayers of porcine aortic endothelial cells exposed for 10 min to 100 microM H2O2 lose their capacity to produce prostaglandins in response to addition of saturating exogenous arachidonic acid. Significant recovery of prostaglandin I2 and E2 synthesis occurred within 3 h and full enzymatic capacity returned by 6 h. Reducing the injury by exposure to half the amount of H2O2 allowed prostaglandin I2 production to recover to a greater extent in 3 h, while cells exposed for 60 min to either 0.5 or 1.0 mM H2O2 demonstrated no recovery. Pre-treatment with either actinomycin D or cycloheximide also prevented recovery following exposure to 100 microM peroxide. Injured cells did not recover when incubated with balanced salts after removal of peroxide, while incubation with medium 199 allowed for the complete return of synthetic capacity. Addition of 1% fetal calf serum in medium 199 did not facilitate recovery. Production of prostaglandins from endogenous arachidonic acid, released by either bradykinin or the ionophore A23187, was also inhibited by H2O2 exposure, however, full recovery of this stimulated synthesis occurred within 3 h. Cycloheximide pre-treatment completely inhibited recovery of bradykinin-induced prostaglandin I2 synthesis. These data demonstrate that sublethal concentrations of H2O2 irreversibly inactivate fatty acid cyclooxygenase and that synthesis of new enzyme is required for recovery. This return of activity occurs more rapidly for production of prostaglandins from endogenous arachidonic acid compared with production following addition of exogenous substrate.

Animals↗

PubMed1988

Repair and recovery in the epithelial and vascular connective tissues of pig skin after irradiation.

Using split-dose experiments, with varying time intervals between two equal fractions, the total repair capacity and the time of onset of additional recovery was determined for both early and late responses in pig skin. The early epidermal response was studied after beta-irradiation and 250 kV X-rays were used to investigate dermal changes. Based on the results of seven separate single dose studies the ED50 value ( +/- SE) for early moist desquamation was 27.76 +/- 0.91 Gy. With intervals of one and 14 days between two equal fractions similar ED50 values of 35 Gy were obtained. This suggested a recovered dose of approximately 7 Gy for epithelial desquamation, a repair capacity for sublethal injury of 20-25%. Additional recovery possibly due to repopulation, was observed with intervals of greater than or equal to 21 days between fractions. The rate of additional recovery was linearly related to the time interval between doses and was equivalent to 74 cGy/day. Recovery from the first dose was complete within 6 weeks. Evidence for radiation-induced tissue hypoxia was obtained when longer time intervals between doses were used. The more subjective early erythema reaction was also assessed. This reaction produced a similar estimate for the repair capacity and for the time of onset of recovery to that obtained using moist desquamation. This agreement was not maintained with intervals of greater than or equal to 28 days between doses due to an artefact associated with the way erythema reactions were assessed. After irradiation with single doses of X-rays ED50 values of 18.59 +/- 0.49 and 20.53 +/- 0.35 Gy were obtained for the dermal reactions of dusky/mauve erythema and necrosis, respectively. The recovered doses for the dermal responses, with intervals of 1 and 28 days between fractions, were similar, approximately 4.2 Gy, indicating a total repair capacity of 20-25%. Additional dermal recovery was seen only with intervals of greater than 28 days between doses. There was no evidence for "slow repair". Surprisingly complete recovery from the first dose was suggested with an interval of 16 weeks between doses. This finding might be influenced by radiation-induced hypoxia. The time of onset of additional repopulation/recovery and the latency for tissue impairment in epidermal and dermal tissues in pig skin were compared with those for other early and late responding tissues.

Animals↗

PubMed1983

Delayed kinetics of recovery of haemopoiesis following autologous bone marrow transplantation. The role of excessively rapid marrow freezing rates after the release of fusion heat.

Thirty-five patients were treated by intensive chemotherapy and/or whole-body irradiation followed by reinjection of cryopreserved autologous bone marrow. In 8 patients the kinetics of recovery of haemopoiesis was delayed (recovery to 10(9) leucocytes/litre beyond day 27 and recovery to 50 X 10(9) platelets/litre beyond day 25). This delay was directly responsible for the death of 3 patients and contributed to a fatal outcome in 2 others (mortality rate 9-14%). Retrospective analysis of these 8 cases revealed that failure of autologous transplantation was associated with poor recovery of CFUc, which was in turn related to an excessively rapid freezing rate after the release of fusion heat. Recovery of CFUc to 50% or more was achieved in 100% of cases when the freezing rate was less than 5 degrees C/min, 45% for freezing rates between 5 and 10 degrees C/min and 22% when the freezing rate exceeded 10 degrees C/min (n = 71, P less than 0.001). There was an inverse linear or logarithmic relationship between CFUc recovery and freezing rate after the transition phase (r = -0.46, r = -0.43, P less than 0.001). The quantity of nitrogen introduced into the freezing chamber to annul the fusion heat must therefore be calibrated with accuracy so that the desired shortening of the transition phase will not be accompanied by an overly marked increase in the freezing rate, which would result in the destruction of stem cells. To ensure an adequate freezing rate, it is crucial to monitor the temperature continuously in each sample of bone marrow during the freezing process. This study also suggested that other factors may have interfered with the kinetics of recovery after autologous bone-marrow transplantation. These factors include myelofibrosis, the presence of an Australia antigen and administration of compounds that are toxic for the bone marrow after reinjection of cryopreserved marrow. However, the responsibility of these factors cannot be stated with certainty.

Adult↗

PubMed1987

The efficiency of growth during body weight recovery in young adult female rats.

1. Six young adult female rats were subjected to 40% body weight loss in 84 days by food restriction followed by recovery of body weight in 36.2 +/- 4.28 days by ad libitum food intake. The regimen was then repeated, with 40% body weight loss in 105 days followed by recovery of body weight in 22.8 +/- 2.00 days. 2. Food intakes were measured continuously. 3. These rats used digestible energy more efficiently during the second recovery (49.1 +/- 3.11 v. 77.3 +/- 7.91 kJ digestible energy intake per g body wt gain). 4. Seven rats were subjected to one body weight loss and recovery. Five of them had an efficiency of 44.1 +/- 3.34 kJ/g live wt gain which was not significantly different from that of the first group during their second recovery; they may not have been capable of improvement after a second deprivation. 5. It is concluded that some rats can adapt after a period of severe body weight loss and recovery to utilize food more efficiently during body weight recovery after a second episode.

Animals↗

PubMed1995

Metabotropic glutamate receptor subtypes differentially influence neuronal recovery from in vitro hypoxia/hypoglycemia in rat hippocampal slices.

Hippocampal slices were transiently exposed to an oxygen- and glucose-free environment which causes a pronounced drop of both ATP and creatine phosphate, an anoxic depolarization, and an incomplete recovery of synaptically evoked population spike in the CA1 region after 1 h (48.5 +/- 3.6% of baseline values). This recovery could be markedly enhanced by the application of N-methyl-D-aspartate receptor antagonists. To examine the influence of metabotropic glutamate receptors on neuronal recovery from hypoxia/hypoglycemia, we applied various antagonists and agonists of the metabotropic glutamate receptors to the bath during the interval from 20 min before to 10 after hypoxia/hypoglycemia. The metabotropic glutamate receptor antagonists (+)-alpha-methyl-4-carboxyphenylglycine and L-2-3- amino-phosphonopropionic acid were both able to enhance the population spike recovery significantly. However, the mixed metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid also exhibited a protective effect on population spike recovery, leaving the anoxic depolarization and N-methyl-D-aspartate responses during the hypoxia/hypoglycemia untouched. With the help of more subtype-specific agonists, we found that an activation of phospholipase C coupled (class 1) metabotropic glutamate receptors prior to hypoxia/hypoglycemia may be responsible for the protective effect seen with 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid, because the specific class 1 metabotropic glutamate receptor agonist trans-azetidine-2,4-dicarboxylic acid appeared to be highly protective, but only if it was applied 20 min before the hypoxia/hypoglycemia. An activation of class 2 metabotropic glutamate receptors by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, which inhibits adenylyl cyclase activity, led to a marked deterioration of the population spike recovery and even to a total prevention of the protective effect of the N-methyl-D-aspartate agonist D-2-amino-5-phosphonopentanoic acid. Our data suggest that prior activation of class 1 metabotropic glutamate receptors is beneficial, while their activation during hypoxia/hypoglycemia is detrimental. Furthermore, the activation of class 2 metabotropic glutamate receptors decreases the recovery from hypoxia/hypoglycemia.

Adenosine Triphosphate↗

PubMedDate not supplied

Behavioral correlates of denervation and reinnervation of the hippocampal formation of the rat: recovery of alternation performance following unilateral entorhinal cortex lesions.

Following unilateral lesions of the entorhinal cortex (E.C.) of the rat, cells in the dentate gyrus which have been deprived of their normal ipsilateral input are reinnervated in part by axons from the contralateral E.C. The proliferation of this crossed projection occurs largely between 8 and 12 days postlesion. The present experiments analyze changes in alternation behavior which occur during this period of afferent proliferation. Rats were trained to alternate responses (L-R) in a T-maze for food reward. Bilateral E.C. lesions resulted in a persistent deficit in alternation performance which did not recover after over 50 days of postoperative testing. Unilateral E.C. lesions, however, resulted in a transient deficit in alternation which recovered over time to preoperative levels. For example, animals permitted a 10-day recovery before the initiation of postlesion testing exhibited no more of a performance deficit than following a 10-day no-training period alone. However, animals permitted only a 3-day postoperative recovery were impaired in the alternation task until 10-12 days postlesion, despite daily training. Thus, recovery of performance following unilateral lesions was dependent on postlesion time rather than the amount of testing/retraining. Since bilateral lesions resulted in a persistent performance deficit while unilateral lesions resulted in a deficit with recovery, we hypothesize that behavioral recovery might be related to the reinnervation of the dentate gyrus by the contralateral E.C. To test this hypothesis, secondary lesions were placed in operated-recovered animals. Secondary lesions of the surviving E.C. resulted in a deficit in alternation performance similar to that following one stage bilateral lesions. In addition, secondary lesions of the dorsal psalterium (the fiber tract which carries the corssed E.C.-dentate projections) also disrupted performance in operated-recovered animals. Primary lesions of the dorsal psalterium alone had only slight and transient effects on alternation performance, however. Thus, the time course of the recovery, the results following bilateral lesions, and the results of secondary lesions are all consistent with the hypothesis that recovery of alternation performance following unilateral E.C. lesions may depend upon the reinnervation of the dentate gyrus by the contralateral E.C.

Afferent Pathways↗

PubMed1992

Behavioural evidence for recovery of auditory function in guinea pigs following kanamycin administration.

Deterioration followed by recovery of behavioural absolute threshold and frequency selectivity has been observed in guinea pigs following kanamycin administration of 200 mg/kg body weight daily for 16 days. Deterioration in function consistently follows a high-to-low frequency pattern and recovery generally occurs at the lowest of the high (8-32 kHz) frequencies affected. The degree of recovery is related to the magnitude of the threshold elevation; where large (40-45 dB) elevations occur initially, the process appears to be partial since threshold recovers only to within 5-12 dB of pre-administration levels. In instances where smaller threshold elevations (5-20 dB) take place initially, recovery can sometimes be complete. However, when threshold elevations of over 50 dB occur, no recovery is apparent. Recovery is relatively slow, taking place over periods of up to 100 days post-kanamycin administration. Hair cell counts have established that the threshold elevation which remains in instances of partial recovery is not related to a reduction in hair cell numbers at the light microscope level.

Acoustic Stimulation↗

PubMed1993

Abnormal systolic blood pressure response during exercise recovery in patients with angina pectoris.

OBJECTIVES: This study was conducted to clarify the mechanisms of the abnormal systolic blood pressure response after exercise in patients with angina pectoris. BACKGROUND: An abnormal systolic blood pressure response in patients with angina pectoris has been observed not only during exercise but also during the recovery period after exercise. However, the mechanisms of this abnormal response during recovery have not been elucidated. METHODS: Thirty-five patients with angina pectoris and 17 control subjects underwent bicycle ergometric studies after insertion of a Swan-Ganz catheter. RESULTS: In control subjects, all hemodynamic variables decreased rapidly after exercise. In 7 of the 35 patients, systolic blood pressure increased after exercise. The patients with angina were classified into two groups. In group I (17 patients), changes in systolic blood pressure during recovery were smaller than those in control subjects. In group II (18 patients) recovery of systolic blood pressure was normal. Changes in stroke index from rest to peak exercise were smaller in group I than in group II. Stroke index in both patient groups increased paradoxically during recovery. The increase in systemic vascular resistance index during recovery and the ratio of plasma norepinephrine concentration to cumulative work load were greater in group I than in group II. CONCLUSIONS: An abnormal systolic blood pressure response after physical exercise in patients with angina pectoris is indicative of severe myocardial ischemia during exercise and may be caused by an increase in stroke volume due to recovery from myocardial ischemia and increased systemic vascular resistance secondary to exaggerated sympathetic nervous activity.

Adult↗

PubMed1995

Recovery from mivacurium-induced neuromuscular blockade after neurosurgical procedures of long duration.

STUDY OBJECTIVE: To determine if recovery following prolonged (5 hours in length or greater) infusions of mivacurium is different from recovery after single bolus administration. DESIGN: open-labelled, controlled study. SETTING: Inpatient neurosurgical service at a university hospital. PATIENTS: 36 patients between the ages of 18 to 65 without significant history of renal, hepatic, cardiac, or metabolic disease undergoing neurosurgical procedures. 21 patients had craniotomies or skull base procedures of an estimated length of 5 hours or greater; 15 patients (control) underwent short neurosurgical operations (two hours or less). INTERVENTIONS: Intravenous (IV) mivacurium 0.15 mg/kg was given with stable general anesthesia with 70% nitrous oxide in oxygen, 0.2% to 0.3% end-tidal isoflurane, and continuous infusion of fentanyl. The control group was allowed to recover spontaneously after single bolus administration while neuromuscular blockade was maintained in the study group with a continuous infusion of mivacurium until 30 minutes before completion of surgery, at which time the infusion was discontinued and neuromuscular function was allowed to recover spontaneously. MEASUREMENTS AND MAIN RESULTS: The evoked compound electromyogram of the adductor pollicis brevis muscle was measured during stimulation of the ulnar nerve at 2 Hz for 2 seconds at 10-second intervals. Measurements included time to 50% and 90% depression of twitch (T1 of the TOF response), time to T1 equal to 25% (T1(25)), 50% (T1(50)), and 75% (T1(75)) of baseline, and TOF ratio (TR) at 10%, 25%, 50%, and 75% recovery. Recovery index (RI), which is T1(75) minus T1(25), was also determined. All mivacurium infusion rates decreased during surgery. Recovery rates were significantly longer in the long infusion (LI) group than the control group. RI was also increased in the LI group compared with the single bolus control (11.3 +/- 1.2 minutes vs. 7.1 +/- 0.8 minutes p < 0.05). CONCLUSIONS: Recovery following mivacurium by prolonged continuous infusion was slower than that observed after single bolus administration in this patient population. Clinically, this increased time to recovery may be insignificant.

Adolescent↗

PubMedDate not supplied

Fusion tails for the recovery and purification of recombinant proteins.

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.

Amino Acids↗

PubMed1993

A method for estimation of extracellular concentration of compounds by microdialysis using urea as an endogenous recovery marker in vitro validation.

A new, practical method called microdialysis has been developed for the estimation of free, unbound drug concentrations in the interstitial fluid. This method is based on the use of urea as an endogenous recovery marker. Urea freely distributes throughout the body water compartment, and its plasma concentration can be used as an accurate measure of its interstitial levels. Microdialysis exploits the relationship between the relative recoveries of urea and the recoveries of an analyte that are established during calibration. The calibration is carried out using different probes, varying perfusion rates, membrane surface areas, temperatures, and tissue models. After obtaining a broad range of recoveries, an empirical mathematical model of the relationship between the recovery of the respective analyte and that of urea is formed. During in vivo experiments, a microdialysis probe is placed into a tissue or fluid of interest, and the urea recovery is monitored by comparing the corresponding dialysate and plasma urea levels. From the calculated urea recoveries, correlation equation, and analyte dialysate concentrations, the extracellular concentration of the unbound analyte in the tissue or fluid can be estimated at any given time point. The purpose of this study is to describe the method of microdialysis and demonstrate its applicability in vitro using two model compounds, theophylline and 3'-azido-3'-deoxythymidine (AZT), in the rat plasma.

Animals↗