PRL3 enhances T-cell acute lymphoblastic leukemia growth through suppressing T-cell signaling pathways and apoptosis.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes and is largely driven by the NOTCH/MYC pathway. Yet, additional oncogenic drivers are required for transformation. Here, we identify protein tyrosine phosphatase type 4 A3 (PRL3) as a collaborating oncogenic driver in T-ALL. PRL3 is expressed in a large fraction of primary human T-ALLs and is commonly co-amplified with MYC. PRL3 also synergized with MYC to initiate early-onset ALL in transgenic zebrafish and was required for human T-ALL growth and maintenance. Mass-spectrometry phosphoproteomic analysis and mechanistic studies uncovered that PRL3 suppresses downstream T-cell phosphorylation signaling pathways, including those modulated by VAV1, and subsequently suppresses apoptosis in leukemia cells. Taken together, our studies have identified new roles for PRL3 as a collaborating oncogenic driver in human T-ALL and suggest that therapeutic targeting of the PRL3 phosphatase will likely be a useful treatment strategy for T-ALL.

Phylogeographical structure and temporal complexity in American sweetgum (Liquidambar styraciflua; Altingiaceae).

Eastern North American plant biogeography has traditionally focused on two primary issues: (i) the location of temperate Pleistocene refugia and their proximity to the southern margin of the ice sheet during the last glacial maximum, and (ii) the origin of the temperate element of northern Latin America. While numerous population genetic and phylogeographical studies have focused on the first issue, few (if any) have considered the second. We addressed these issues by surveying 117 individuals from 24 populations of Liquidambar styraciflua (American sweetgum; Altingiaceae) across the southeastern USA, eastern Mexico, and Guatemala, using more than 2200 bp of chloroplast DNA sequence data. To specifically address the issue of timing, we estimated intraspecific divergence times on the basis of multiple fossil-based calibration points, using taxa from Altingiaceae (Liquidambar and Altingia) and Hammamelidaceae (Hamamelis) as outgroups. More than half of the sampled localities exhibited multiple haplotypes. Remarkably, the greatest variation was observed within the USA, with Mexico and Guatemala sharing widespread haplotypes with Texas, Mississippi, Kentucky, Ohio, and northern Virginia. This lack of differentiation suggests shared ancestral polymorphisms, and that the genetic signal we observed is older than the disjunction itself. Our data provide support for previously proposed hypotheses of Pleistocene refugia in peninsular Florida and along the eastern Atlantic, but also for deeper divergences (approximately 8 million years ago) within the USA. These patterns reflect a dynamic biogeographical history for eastern North American trees, and emphasize the importance of the inclusion of a temporal component in any phylogeographical study.

Autosomal dominant canine malignant hyperthermia is caused by a mutation in the gene encoding the skeletal muscle calcium release channel (RYR1).

BACKGROUND: Malignant hyperthermia (MH) is an inherited disorder of skeletal muscle characterized by hypercarbia, rhabdomyolysis, generalized skeletal muscle contracture, cardiac dysrhythmia, and renal failure, that develops on exposure to succinylcholine or volatile anesthetic agents. All swine and up to 50% of human MH events are thought to be associated with mutations in the calcium release channel of the sarcoplasmic reticulum, also known as the ryanodine receptor (RYR1). Events resembling MH have been reported in other species, but none have undergone genetic investigation to date. METHODS: To determine the molecular basis of canine MH, a breeding colony was established with a male, mixed-breed, MH-susceptible (MHS) dog that survived an in vivo halothane-succinylcholine challenge. He was mated to three unaffected females to produce four litters and back-crossed to an affected daughter to produce one litter. One of his MHS sons was mated to an unaffected female to produce an additional litter. Forty-seven dogs were phenotyped with an in vitro contracture test and diagnosed as MHS or MH normal based on the North American in vitro contracture test protocol. Nine microsatellite markers in the vicinity of RYR1 on canine chromosome 1 (CFA01) were tested for linkage to the MHS phenotype. Mutational analysis in two MHS and two MH-normal dogs was performed with direct sequencing of polymerase chain reaction products and of cloned fragments that represent frequently mutated human RYR1 regions. A restriction fragment length polymorphism was chosen to detect the candidate mutation in the pedigree at large. RESULTS: Pedigree inspection revealed that MHS in this colony is transmitted as an autosomal dominant trait. FH2294, the marker closest to RYR1, is linked to MHS at a theta = 0.03 with a LOD score of 9.24. A T1640C mutation gives rise to an alanine for valine substitution of amino acid 547 in the RYR1 protein, generating a maximum LOD score of 12.29 at theta = 0.00. All dogs diagnosed as MHS by in vitro contracture test were heterozygous for the mutation, and all MH-normal dogs were homozygous for the T1640 allele. CONCLUSIONS: These results indicate that autosomal dominant canine MH is caused by a mutation in the gene encoding the skeletal muscle calcium release channel and that the MHS trait in this pedigree of mixed-breed dogs is in perfect cosegregation with the RYR1 V547A mutation.

A technique for evaluating analgesia in conscious perching birds.

OBJECTIVE: To develop a technique for objective assessment of modulation of nociperception in conscious perching birds. ANIMALS: 31 adult African grey parrots. PROCEDURE: Birds were randomly assigned to receive saline (0.9% NaCl) solution (n = 10), butorphanol tartrate (11), or buprenorphine hydrochloride (10), i.m. Birds were fitted with a surface electrode on the medial metatarsus of 1 leg. An electrical stimulus was delivered to the bird's foot through an aluminum surface on half of the perch. The alternate side of the perch delivered a noxious thermal stimulus. A withdrawal response to either stimulus was recorded when the bird lifted its foot or vigorously flinched its wings. RESULTS: Responses to thermal stimuli were extremely variable during baseline testing and after administration of drugs. Thus, significant differences were not detected after drug injection. In contrast, responses to an electrical stimulus were predictable with much less variation. CONCLUSIONS AND CLINICAL RELEVANCE: This method and device allowed for the reliable determination of withdrawal threshold in perching birds. Use of this technique for objective assessment of modulation of nociperception in conscious perching birds will enable assessment of analgesic drugs.

Analgesic effects of butorphanol and buprenorphine in conscious African grey parrots (Psittacus erithacus erithacus and Psittacus erithacus timneh).

OBJECTIVE: To evaluate effects of butorphanol tartrate and buprenorphine hydrochloride on withdrawal threshold to a noxious stimulus in conscious African grey parrots. ANIMALS: 29 African grey parrots (Psittacus erithacus erithacus and Psittacus erithacus timneh). PROCEDURE: Birds were fitted with an electrode on the medial metatarsal region of the right leg, placed into a test box, and allowed to acclimate. An electrical stimulus (range, 0.0 to 1.46 mA) was delivered to each bird's foot through an aluminum perch. A withdrawal response was recorded when the bird lifted its foot from the perch or vigorously flinched its wings. Baseline threshold to a noxious electrical stimulus was determined. Birds then were randomly assigned to receive an i.m. injection of saline (0.9% NaCl) solution, butorphanol (1.0 mg/kg of body weight), or buprenorphine (0.1 mg/kg), and threshold values were determined again. RESULTS: Butorphanol significantly increased threshold value, but saline solution or buprenorphine did not significantly change threshold values. CONCLUSIONS AND CLINICAL RELEVANCE: Butorphanol had an analgesic effect, significantly increasing the threshold to electrical stimuli in African grey parrots. Buprenorphine at the dosage used did not change the threshold to electrical stimulus. Butorphanol provided an analgesic response in half of the birds tested. Butorphanol would be expected to provide analgesia to African grey parrots in a clinical setting.

Comparison of isoflurane with sevoflurane for anesthesia induction and recovery in adult dogs.

OBJECTIVE: To compare mask anesthesia induction and recovery characteristics between 2 inhalant anesthetic agents: isoflurane and sevoflurane. ANIMALS: 16 clinically normal, young adult Beagles. PROCEDURE: Using a cross-over design, dogs were randomly selected to receive sevoflurane or isoflurane via a face mask and a circle anesthetic system. Vaporizer setting concentrations were increased in stepwise, equal minimum alveolar concentrations (MAC) for each anesthetic until the vaporizer setting of 2.6% for isoflurane or 4.8% for sevoflurane (2 MAC) was reached. Concentration was kept constant until the dog had a negative tail clamp response and was intubated. End-tidal concentration was maintained at 1.8 to 2.0% or 3.3 to 3.8% for isoflurane or sevoflurane, respectively (1.4 to 1.6 MAC) for 30 minutes. Dogs were allowed to recover with only tail clamp stimulation until a positive response was obtained. Extubation was performed when a spontaneous swallow reflex was observed. Dogs were allowed to achieve sternal recumbency and stand unassisted without further stimulation. RESULTS: Sevoflurane induction resulted in shorter time to loss of palpebral reflex, negative tail clamp response, and time to tracheal intubation, and was of better quality than isoflurane induction. Both anesthetics were associated with rapid and smooth recovery. CONCLUSIONS: Sevoflurane mask induction is faster and of better quality, compared with isoflurane, in adult dogs. Recovery time and quality are comparable. CLINICAL RELEVANCE: On the basis of these results, sevoflurane is a suitable inhalant anesthetic for mask induction and recovery in adult dogs and appears to have some advantages over isoflurane, including faster and smoother mask induction.

Effects of mydriatic agents in cockatoos, African gray parrots, and Blue-fronted Amazon parrots.

OBJECTIVE: To compare, in psittacines, the mydriatic effects of several topically applied curariform, sympathomimetic, and parasympatholytic drugs with and without the addition of surface-acting penetrating agents. DESIGN: Prospective, randomized controlled trial. ANIMALS: 10 adult cockatoos (Cacatua sulphurea subspecies), 2 adult African gray parrots (Psittacus erithacus), and 3 adult Blue-fronted Amazon parrots (Amazona aestiva). PROCEDURE: Three curariform drugs (d-tubocurarine, pancuronium, and vecuronium bromide) and 2 autonomic drugs (atropine and phenylephrine hydrochloride) were evaluated. Drugs were tested with and without the addition of a surface-acting penetrating agent, either saponin or benzalkonium chloride. The agent that resulted in the most significant change in pupillary diameter with the fewest systemic side effects in the cockatoos then was evaluated for its effects in the African gray parrots and the Blue-fronted Amazon parrots. During each drug trial, 1 eye was randomly selected to receive the control drug (0.9% NaCl), and the opposite eye was selected to receive the test drug. Each pupil was videotaped 5 (cockatoos only), 15, 30, 45, 60, and 75 minutes after treatment. Pupil diameters were measured by use of a computerized image analysis system. Data for pupil size were analyzed by means of repeated measures ANOVA. RESULTS: Vecuronium without the addition of a surface-acting penetrating agent produced the most consistent and greatest pupillary dilatation in all 3 species with the fewest systemic side effects. CLINICAL IMPLICATIONS: Vecuronium is potentially a clinically useful, topical mydriatic agent for use in avian species. Documented differences in the prevalence of systemic side effects between species suggests that caution should be applied when applying this drug bilaterally.

Determination of the ED50 of isoflurane and evaluation of the isoflurane-sparing effect of butorphanol in cockatoos (Cacatua spp.).

The use of butorphanol as an analgesic in a psittacine species was evaluated by determining its isoflurane-sparing effects. The Effective Dose 50 (ED50) of isoflurane was determined using a bracketing technique based on the purposeful movement elicited by pressure applied to a digit with a hemostat. The ED50 of isoflurane for 11 cockatoos (four greater sulfur crested, three lesser sulfur crested, and four citron crested) was determined to be 1.44 +/- 0.07%. After the administration of 1 mg/kg of butorphanol tartrate intramuscularly (IM), the ED50 was significantly (P < .05) decreased to 1.08 +/- 0.05%. Physiological variables that changed significantly included decreases in heart rate, tidal volume (Vt), inspiratory (Ti) and expiratory times (Te), and an increase in respiratory rate. No complications resulted because of these changes. Based on the results, butorphanol produces an isoflurane-sparing effect in cockatoos and has the potential to be a useful analgesic in psittacines.

Barotrauma in a cat.

A 3.5-year-old domestic long-hair cat was admitted to the veterinary hospital for routine procedures, including dental prophylaxis. The cat appeared clinically normal. General anesthesia was induced, and 30 minutes later, the pop-off valve was inadvertently left in a closed position. The cat developed pneumothorax, which was treated via thoracentesis and administration of oxygen. The condition resolved and the cat was released from the hospital to its owner. Barotrauma resulted because of high pressure in the anesthetic circuit. Barotrauma is a life-threatening complication of general anesthesia.

Parenteral anticholinergics in dogs with normal and elevated intraocular pressure.

Dogs given parenteral anticholinergic drugs have been thought to be at risk for development or exacerbation of elevated intraocular pressure (IOP). In a randomized, blinded, placebo-controlled study, we evaluated the effect of intramuscular glycopyrrolate (0.01 mg/kg) on pupil diameter and IOP in unanesthetized normal dogs. Treatment with glycopyrrolate did not change pupil diameter or IOP from baseline, nor were there differences between glycopyrrolate and saline-treated (control) dogs. In addition, the authors retrospectively reviewed the medical records of 2,828 dogs undergoing general anesthesia between April 1987 and September 1990 to determine if there was an association between parenteral anticholinergic medication and postanesthetic elevation in IOP. The authors also determined the frequency of bradycardia requiring anticholinergic therapy during anesthesia in dogs with glaucoma. Of the 2,828 cases reviewed, the records of 46 dogs coded for glaucoma were examined in detail. The 46 dogs underwent 62 episodes of anesthesia, with 23 episodes including exposure to an anticholinergic drug. An increase in IOP from preanesthetic to postanesthetic measurement occurred in three dogs. One of these dogs received anticholinergic medication for bradycardia during anesthesia. The postanesthetic elevation in IOP in this dog was probably not drug related. Preanesthetic anticholinergic administration did not affect the incidence of anticholinergic administration for bradycardia during the anesthetic episode. Anticholinergic therapy during anesthesia was more frequent when the preanesthetic medication included an opiate drug. These studies do not indicate an association between parenteral anticholinergic administration and elevations in IOP.

DNA damage and mutation in human cells exposed to nitric oxide in vitro.

Nitric oxide (NO.) is a physiological messenger formed by several cell types. Reaction with O2 forms oxides that nitrosate amines at pH values near 7. We now report experiments in which NO. was added to intact human cells and to aerobic solutions of DNA, RNA, guanine, or adenine. TK6 human lymphoblastoid cells were mutated 15- to 18-fold above background levels at both the HPRT and TK gene loci. Xanthine and hypoxanthine, from deamination of guanine and adenine, respectively, were formed in all cases. NO. induced dose-responsive DNA strand breakage. Yields of xanthine ranged from nearly equal to about 80-fold higher than those of hypoxanthine. Yields of xanthine and hypoxanthine from nucleic acids were higher than those from free guanine and adenine. This was most pronounced for xanthine; 0.3 nmol/mg was formed from free guanine vs. 550 nmol/mg from calf thymus RNA. Nitric oxide added to TK6 cells produced a 40- to 50-fold increase in hypoxanthine and xanthine in cellular DNA. We believe that these results, plus the expected deaminations of cytosine to uracil and 5-methylcytosine to thymine, account for the mutagenicity of nitric oxide toward bacteria and mammalian cells.

Use of halothane and isoflurane in the horse.

When compared with halothane, isoflurane has several distinct characteristics. Vaporizer settings are higher because of its lower potency. Respiratory rates will be slower, and intraoperative changes in depth and recovery from surgical depth of anesthesia will be more rapid, although total recovery times frequently will not be different. Halothane and isoflurane appear similar in their effects on ocular reflexes and mean arterial blood pressure. Recovery from isoflurane should be managed to provide added sedation or physical support if the horse attempts to stand prematurely.

Doxacurium and mivacurium do not trigger malignant hyperthermia in susceptible swine.

The role of succinylcholine in the precipitation of malignant hyperthermia (MH) necessitates the testing of new neuromuscular relaxants for their ability to trigger MH in MH-susceptible swine before general human use. We tested doxacurium and mivacurium, two new nondepolarizing bis-benzylisoquinolinium neuromuscular relaxants, at ED95 and at four times ED95 doses in swine previously documented to be MH-susceptible. In none of the 16 animals was MH triggered after administration of these relaxants, whereas all animals developed fatal MH after administration of halothane or halothane plus succinylcholine. Muscle biopsy specimens taken before administration of the relaxant confirmed that all animals had increased sensitivity to halothane, caffeine, or both. Thus, we conclude that doxacurium and mivacurium are not triggering agents of malignant hyperthermia in MH-susceptible swine.

Cardiovascular and respiratory effects of inspired oxygen fraction in halothane-anesthetized horses.

Anesthesia of equids is associated with pulmonary dysfunction. Cardiovascular and respiratory effects of inhalation anesthetic agents and duration of anesthesia have been studied, using oxygen as the carrier gas. To our knowledge, the effects of inspired oxygen have not been determined. We studied the cardiovascular and respiratory effects of 2 inspired oxygen fractions (0.30 and greater than 0.85) in 5 laterally recumbent, halothane-anesthetized horses. Mean systemic arterial blood pressure, cardiac output, central venous pressure, pulmonary arterial pressure, arterial pH, and arterial base excess were similar in horses of the 2 groups during 4 hours of anesthesia at constant end-tidal halothane concentration. End-tidal partial pressure of CO2, arterial partial pressure of CO2 and O2, and alveolar-to-arterial O2 tension difference were greater in horses exposed to the higher oxygen concentration. On the basis of the data obtained, we suggest that greater hypoventilation and ventilation/perfusion mismatch occur when horses are breathing high-oxygen fraction. Arterial partial pressure of O2 was not different between the 2 groups of horses after they were disconnected from the anesthesia circuit and allowed to breathe room air. Horses recovered from anesthesia without complications.

Bonding to resurfaced posterior composites.

Despite significant improvements in the wear resistance of posterior composite restorations, they still undergo occlusal wear, color change, and surface staining with time. One method of repairing these restorations is re-surfacing the old composite. This study investigated the bonding of new composite to the corresponding old composite material by several different mechanical conditioning steps, chemical conditioning steps, primer conditioning steps, and four posterior composites. Aged composite surfaces were conditioned, re-bonded to new composite, stored in artificial saliva for seven days at 37 degrees C, and tested in shear. The mean shear strength for unrepaired composites was 27 MPa. Optimal re-bond strengths were 88% (Estilux), 77% (Ful-Fil), 92% (Occlusin), and 102% (P-10) of original bulk shear strengths. General linear modeling revealed that the best combination tested was roughening with a D830 diamond bur, conditioning the surface with water, and priming with Scotchbond-1 dentin bonding agent.

Induction of equine postanesthetic myositis after halothane-induced hypotension.

Wick catheters were used to measure intracompartmental pressures of the extensor carpi radialis muscles and long heads of the triceps brachii muscles of 7 horses maintained under halothane anesthesia during controlled ventilation. Horses were positioned in left lateral recumbency on a water bed for 4 hours. Using a crossover design, 6 of the 7 horses were subjected to normotensive and hypotensive anesthesia on separate occasions. Hypotension was achieved by increasing the inspired halothane concentration. Hematologic and biochemical measurements were determined at designated intervals before, during, and for 7 days after each anesthetic episode. Under hypotensive conditions, 2 horses developed severe generalized myositis and were euthanatized. Three of the 5 other horses developed swelling of the downside masseter muscle, 4 demonstrated mild extensor deficits of the downside forelimb, and 1 had a severe extensor deficit of the uppermost hind limb. As a group, the hypotensive horses had markedly increased activities of serum enzymes (creatine kinase, aspartate transaminase, and blood lactate) and abnormalities in calcium-phosphorus homeostasis. Lameness or enzyme alterations were not observed in normotensive horses. Although the intracompartmental pressure values were markedly increased in the muscle bellies of the compressed limbs of all horses, there was a statistically significant difference in intracompartmental pressures between the downside or compressed muscle compartments of the extensor carpi radialis of hypotensive and normotensive horses. High concentrations of halothane may predispose anesthetized horses to postanesthetic myositis, even when protective padding is used. Intracompartmental muscle pressure, as measured by the wick catheter, may not be a reliable predictor of equine postanesthetic lameness.

Dental dolorimetry for the evaluation of an analgesic agent in the horse.

A monopolar electrode was implanted surgically in the canine tooth dentine layer to evaluate pain threshold responses of horses. A constant-current stimulator was used to deliver a known electrical current to the tooth pulp nerve. A single stimulus of 2-ms duration, repeated at greater than or equal to 20-s intervals, was used to elicit a head lift response. The lowest current level that produced 3 positive head lift responses was recorded as the pain threshold of the horse. The testing technique, dental dolorimetry, was easily performed. Tooth pulp pain thresholds (TPPT) were established on 8 nonmedicated adult male horses. Electrodes were nonreactive and remained functional for up to 98 days. Base-line TPPT values were consistent with repeated measurements on the same day and measurements on subsequent test days. The quantity of electrical current necessary to elicit the TPPT was increased after administration of xylazine HCl as a test analgesic.

Comparative analgesia of xylazine, xylazine/morphine, xylazine/butorphanol, and xylazine/nalbuphine in the horse, using dental dolorimetry.

Xylazine, morphine, butorphanol, and nalbuphine were evaluated in 5 adult male horses, using dental dolorimetry. Comparisons were made at 30, 60, and 100 minutes after IV drug administration. Peak analgesia and the time to develop peak analgesia also were compared. Xylazine induced a marked increase in the tooth pulp pain threshold measurements as did the xylazine/narcotic combinations. Statistical differences were not detectable between these treatments. Xylazine and xylazine/butorphanol were better analgesics than was butorphanol alone at 30 and 60 minutes. Xylazine resulted in peak analgesia faster than did butorphanol or the combination of xylazine/butorphanol. Additive analgesic effects were not detected with the combined treatments.

Differential effect of ketamine on cholinergic- and noncholinergic-induced contractions of isolated guinea-pig bronchi.

Ketamine, 3 X 10(-4) M, was found to inhibit transmural electrical stimulation-induced contractile responses of the mainstem and hilar bronchi isolated from reserpine-pretreated guinea-pigs. This concentration of ketamine did not cause direct smooth muscle relaxation in these preparations and a smaller concentration (3 X 10(-5) M) was ineffective in blocking contractions. The pattern of inhibition produced by ketamine was similar to that produced by atropine, 1 X 10(-6) M, in both preparations and was observed at the larger stimulation frequencies employed. When studied in the presence of atropine to optimize noncholinergic-induced contractions, ketamine did not antagonize contractile responses to transmural electrical stimulation. After pretreatment of the isolated tissues with capsaicin, 1 X 10(-6) M, to optimize cholinergic-induced contractions, ketamine produced inhibition of responses to electrical stimulation. Ketamine, 3 X 10(-4) M, produced a shift to the right of the dose-response curve without altering the maximum contractile responses to carbachol, but had no effect on dose-response curves to substance P, the putative noncholinergic transmitter in these bronchial segments. The data suggest that ketamine exerts a selective inhibitory effect on cholinergically mediated contractions in guinea-pig bronchi and that this antagonism is largely exerted post-junctionally, at the level of the smooth muscle muscarinic receptors.