Effects of polyethylene glycol on reverse transcriptase and other polymerase activities.

Polyethylene glycol enhances reverse transcription, augmenting both the rate and duration of polymerization. The effective mean molecular weight of polyethylene glycol is 6000 and the optimal concentration is 12% (w/w). Polyethylene glycol is effective on the reverse transcriptase reaction of all ten type B, C, and D viruses tested under a variety of exogenous, endogenous, and reconstitution assay systems, including the highly efficient conditions involving calf thymus DNA oligonucleotide primers. By three methods of synthesis, polyethylene glycol increased the yields of complementary [3H]DNA by a factor of 1.8--6.5. Polyethylene glycol does not alter the divalent cation requirements of the specificities of the enzyme. Complementary [3H]DNAs made in the presence of polyethylene glycol are indistinguishable in terms of size and sequence complementarity from those made in the absence of the polymer. The stimulatory effect was partly due to the ability of polyethylene glycol to stabilize reverse transcriptase. Preliminary tests indicate that polyethylene glycol also stimulates other nucleotide polymerases, such as the DNA-dependent DNA and RNA polymerases of Escherichia coli and the terminal transferase of calf thymus.

Uptake of Tc-99m MAA by the liver during a thromboscintigram/lung scan.

A young man with a swollen left leg was referred to us for a thromboscintigram/lung scan. The unexpected visualization of the liver during the flow study after intravenous injection of Tc-99m MAA into the dorsal veins of both feet provided the clue to his underlying problem. He was shown to have extensive venous collateralization in the left pelvis, and to a lesser degree on the right, due to a large pelvic mass. This was shown to consist of metastases from a previously treated testicular carcinoma. Venous drainage from the legs was shunted into the mesenteric circulation, which them emptied into the portal vein, thereby carrying the radiopharmaceutical to the liver. No uptake was seen in the spleen.

Sensitivity of mononuclear leucocytes to glucocorticoids in elderly hip-fracture patients resistant to suppression of plasma cortisol by dexamethasone.

OBJECTIVE: Elderly women with proximal femur fracture show a prolonged increase in plasma cortisol, which could have undesirable catabolic effects. Suppression of cortisol by dexamethasone is impaired, suggesting resistance to glucocorticoid effects at feedback inhibitory sites. We therefore wished to find out whether peripheral glucocorticoid sensitivity is normal. DESIGN: Peripheral blood mononuclear leucocytes were used as a model tissue. Blood samples were taken from elderly women about 2 weeks after hip fracture and from elderly control women. Each patient was then given 1 mg dexamethasone at 2300 h followed by further sampling at 0800 and 1600 h the next day. METHODS: Glucocorticoid-receptor binding parameters were measured by incubating whole cells with [3H]dexamethasone for 2 h at 37 degrees C. Inhibition of cell proliferation by dexamethasone was assessed by addition of [3H]thymidine to cells cultured for 65 h with concanavalin A. Cortisol and dexamethasone concentrations were measured in the dexamethasone suppression test. RESULTS: As expected, the hip-fracture patients had raised morning cortisol concentrations and impaired suppression by dexamethasone. The cells of the patients had similar numbers of glucocorticoid receptors to those of the control subjects but higher values for Kd (i.e. a lower binding affinity). The cells of the patients incorporated less [3H]thymidine than the control cells in the absence of dexamethasone. The percentage inhibition by a saturating concentration of dexamethasone was unchanged but the concentration giving half-maximal inhibition was decreased (sensitivity was increased) at the higher of the two concanavalin A concentrations used. CONCLUSIONS: These experiments in mononuclear leucocytes give no evidence of peripheral resistance to glucocorticoids in hip-fracture patients with impaired suppression of cortisol by dexamethasone.

Uptake of radiolabeled leukocytes in prosthetic graft infection.

The utility of radionuclide labeled leukocytes in the demonstration of infection within vascular prostheses was examined. The infrarenal aorta was replaced with a 3 cm Dacron graft in 12 dogs. On the third postoperative day, six of the animals received an intravenous injection of 10(8) Staphylococcus aureus. Labeled leukocyte scans were performed at postoperative days one and three, and then weekly for 8 weeks with indium-111 and technetium-99 labeled autologous leukocytes. When scans showed focal uptake of isotope in the area of prosthetic material, the grafts were aseptically excised and cultured on mannitol-salt agar. Both control and infected animals had retroperitoneal isotope activity in the immediate postoperative period that disappeared by the end of the first week. By the eighth postoperative week, all of the animals that received the bacteremic challenge had both radionuclide concentration in the region of the vascular prosthesis and S. aureus cultured subsequently from the perigraft tissues. None of the control animals had either radionuclide or bacteriologic evidence of infection at the eighth postoperative week. The radiolabeled leukocyte scan is a highly sensitive and specific technique, clinically applicable for the diagnosis of vascular prosthetic infections.

Nuclear medicine in diagnosis and treatment of diseases of the head and neck: II.

The advent of both improved imaging systems and new radioactive agents has increased the effectiveness of nuclear medicine in diagnosing and treating diseases of the head and neck. In this second of two articles, we discuss radionuclide bone imaging and the role of nuclear medicine in the management of thyroid disease. Radionuclide bone imaging is useful in the differential diagnosis of sinusitis, the early detection of head and neck fracture, the assessment of temporomandibular joint disease, and the identification of local extension of primary head and neck carcinoma. In the management of thyroid disease, radionuclide technology is uniquely helpful in the diagnostic evaluation of the thyroid nodule and radioactive iodine continues to play a major role in thyroid cancer therapy.

Nuclear medicine in diagnosis and treatment of diseases of the head and neck. I. Salivary and parathyroid gland disease and one identification and staging of head and neck tumors.

The advent of both improved imaging systems and new radioactive agents has increased the effectiveness of nuclear medicine in diagnosing and treating diseases of the head and neck. In this first in a series of two articles, the role of nuclear medicine is discussed in the evaluation of diseases of the salivary and parathyroid glands, and in the identification and staging of head and neck tumors. Radionuclide studies of the salivary glands are useful in the identification of tumors and the evaluation of gland function. Such studies are a valuable adjunct in the diagnosis of Sjögren's syndrome and of acute and chronic inflammatory disease. Radionuclide imaging also has been helpful in the detection of adenomata and hyperplasia of the parathyroid glands and often complements ultrasonography localization procedures. The advent of gallium-67 imaging has improved the staging of head and neck tumors.

Cortisol kinetics in elderly women with persistently high cortisol concentrations after proximal femur fracture.

OBJECTIVES: Elderly women with proximal femur fracture show abnormal persistence of increased cortisol concentrations, which could contribute to the high morbidity associated with this injury. Two weeks after injury, the authors found substantially increased urinary free cortisol excretion, which usually reflects the integrated concentration of free (bioactive) cortisol in plasma. However, there was a proportionally smaller increase in cortisol production rate. The authors have now tested the hypothesis that this was caused by a decreased metabolic clearance rate (MCR) rather than increased renal clearance, because the latter but not the former would invalidate free cortisol excretion as an index. SETTING: Orthopaedic wards in a teaching hospital. PATIENTS: Thirteen women aged seventy-one to ninety-two years who had sustained a proximal femur fracture approximately two weeks earlier were compared with ten healthy women aged sixty-seven to eighty-three years. These subjects are similar to those in the authors' previous study. MAIN OUTCOME MEASUREMENTS: The authors used single injections of [3H] cortisol to measure its MCR and estimated hepatic blood flow with indocyanine green. RESULTS: The patients with hip fractures had higher plasma cortisol concentrations than did the healthy subjects, as expected. Cortisol MCR was approximately 20 percent lower in the patients, and estimated hepatic blood flow was approximately 35 percent lower in the patients. Analysis of covariance showed that the difference in MCR was the result of the small difference in age between the groups rather than to injury per se. CONCLUSIONS: A lower cortisol MCR in the patients with hip fractures explains the authors' previous results and validates urinary free cortisol excretion as an index. The data suggest a roughly threefold mean increase in plasma cortisol bioactivity two weeks after hip fracture.

The effect of aging on the metabolic clearance rate and distribution of cortisol in man.

Previous studies of cortisol kinetics in old people have been flawed. All but one used a large dose of unlabelled cortisol, which will itself alter the kinetic parameters, and in none was metabolic clearance rate (MCR) calculated. We have, therefore, injected [(3)H]cortisol into men aged 20-38 and healthy (screened) men and women aged 63-83 years and followed its disappearance from the circulation for 3 h. In all three groups the disappearance curves corresponded closely to a double exponential, with half-lives of around 5 and 65 min. A two-pool model was assumed, one being purely a side-pool. The initial and total volumes of distribution and the MCR, but not the clearance rate for exchange between the two pools, tended to be lower in the elderly men than in the young; only the difference in total volume was significant. All these parameters were lower in the elderly women than in the elderly men. We conclude that any decline in cortisol MCR and related kinetic parameters with aging in men is small compared with variation from other sources. These parameters are lower in elderly women than men, in line with a reported sex difference in MCR in young subjects.

The effects of stress and injury on the activity of phosphoenolpyruvate carboxykinase in the liver of the rat.

The effects of stress (diethyl ether anaesthesia for 4-8 min, or intravenous injection of 0.05 ml of a dimethyl sulphoxide/water mixture) and of a scald injury given under ether anaesthesia on hepatic PEPCK (phosphoenolpyruvate carboxykinase, EC 4.1.1.32) were studied in the post-absorptive rat. Injury raised PEPCK activity by about 70% in 2 h and by over 100% in 4 h, over three times as fast as in animals that had only been handled (controls). The two stresses, both of types commonly imposed in animal experiments, had almost as much effect as injury for the first 2 h, although much less thereafter. The roles of sympathetic stimulation and corticosterone in mediating these rises were studied by using alpha beta-blockers and trilostane respectively as inhibitors. (Trilostane only decreased corticosterone concentrations to a little above control values.) The ether-induced increase was somewhat decreased by alpha beta-blockade, but was only eliminated by combined alpha beta-blockade and trilostane. After injury, however, PEPCK synthesis was unaffected by either alpha beta-blockade or trilostane, although it was decreased by their combined action; and it seems that either corticosterone or sympathetic stimulation was sufficient to stimulate PEPCK synthesis maximally. Stimulation by corticosterone was much greater than reported previously by others, for reasons that are discussed. Sympathetic stimulation may have been mediated by glucagon and cyclic AMP, since injury raised portal glucagon concentrations, and stress and injury raised those of hepatic cyclic AMP. PEPCK synthesis was, however, stimulated despite increases in portal insulin concentration, and was not related to the [insulin]/[glucagon] ratio. Thus stress and injury over-rode normal control mechanisms.

[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate.

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of glucose and [14C]glucose between erythrocytes and plasma in the rat.

1. Of the glucose in rat blood 79.8+/-3.3% (s.d.) was in the plasma. The variance was mostly due to differences between rats. 2. The concentration of glucose in erythrocyte water was 51+/-8% (s.d.) of that in plasma water. 3. The ratio (specific radioactivity in plasma)/(specific radioactivity in whole blood), i.e. the P/B ratio, was estimated for glucose at intervals after intravenous injection of [U-(14)C]glucose and [U-(14)C]fructose. The ratio differed from unity by more than the standard error of a single determination of the specific radioactivity of blood or plasma glucose except from 10 to 17min. after injection of [(14)C]glucose and from 22 to 30min. after injection of [(14)C]fructose. At all other times specific radioactivities in blood had to be corrected to give specific radioactivities in plasma. How to do so is described. 4. The P/B ratios were accounted for by a turnover of glucose in erythrocytes of 0.14mumole/min./ml. of erythrocytes. 5. Metabolism of glucose in rat erythrocytes is unlikely to be a major source of lactate.

The recovery of function of chronically obstructed and infected ureters.

Twelve experiments in which intraluminal ureteral pressure, ureteral diameter, aortic blood pressure, and respirations were measured, and in which ureteral wall tension was calculated, were performed on 11 female dogs. These experiments demonstrated that the impairment in ureteral function caused by chronic obstruction and infection could be reversed after surgical correction of the obstruction in conjunction with antibiotic therapy.

Brown adipose tissue activity and oxygen consumption after scald injury in the rat.

Scald injury (30% surface scald) in the rat caused rapid (1-3 h) and transient decreases in oxygen consumption (VO2 20%), colonic temperature (1.1 degree C), and brown adipose tissue (BAT) thermogenic activity (22%). Three days after injury, VO2 was slightly increased in injured rats, and sympathetically mediated heat production (assessed from the inhibitory effect of a beta-adrenergic antagonist on VO2) was significantly greater than than for controls. At this time, BAT activity (in vitro mitochondrial GDP binding) was 35% higher in injured than control rats. Food intake was inhibited for only 24 h in injured animals, but weight gain was suppressed for at least 3 days. The data indicate that sympathetic modification of BAT thermogenesis may contribute to the changes in metabolic rate and body weight gain after scald injury in the rat.

Effects of halothane on glucose metabolism after injury in the rat.

The effects of halothane anaesthesia on glucose metabolism have been investigated in rats after a non-lethal scald injury. Anaesthesia was induced about 70 min after injury. Glucose metabolism was studied at two stages: during and shortly after induction, and about 2 h after induction. Comparisons were made with conscious rats at the corresponding times after injury. All rats were in an ambient temperature of 30 degrees C. During and shortly after the induction of anaesthesia, halothane caused a rapid increase in plasma glucose concentration, which by 30 min had begun to return to the values in injured controls; thus glucose production and utilization were increased. Insulin concentrations were increased also. However, after 2 h exposure halothane had decreased glucose production and utilization, as determined with [5-3H]- and [U-14C]-glucose, increased plasma concentrations of insulin and decreased liver concentrations of glycogen, that is it had exacerbated well-known effects of injury in the rat, including insulin resistance. Hyperglycaemia was not increased.

Recent developments in employee benefits.

The latter part of 1996 and the first part of 1997 produced continuing changes to the laws concerning employee benefits, as both Congress and the courts continued to focus on this area of the law. This article highlights some of the more important developments during the period with particular focus upon those of concern to the insurance industry.

Rates of glucose utilization and glucogenesis in rats in the basal state induced by halothane anaesthesia.

1. Rates and rate coefficients of glucose utilization and replacement were determined with [5-3H]- and [U-14C]-glucose in rats starved for 24h, either conscious or under halothane anaesthesia, in a thermoneutral environment. Plasma insulin concentrations were also measured. 2. Halothane anaesthesia decreased the turnover rate by 20%, which was similar to previously reported decreases in metabolic rates caused by natural sleep. 3. Fractional recycling of glucose carbon was little affected by halothane. 4. Comparison of values in one rat with those in another, among both conscious rats and those under halothane anaesthesia, showed that rate coefficients were inversely correlated with plasma glucose concentrations. 5. These findings indicated that halothane, in the concentration used (1.25%, v/v), had little specific effect on glucose metabolism. 6. Although equilibrium plasma glucose concentrations in different rats under halothane were widely different (4-8 mmol/l) the rates of utilization were very similar (2.5-3.1 micronmol/min per 100 g), indicating that these rates were determined by the production of glucose from gluconeogenic precursors released by basal metabolism, the rate of which is necessarily similar in different rats. 7. Among rats under halothane anaesthesia plasma insulin concentrations were negatively correlated with rate coefficients, showing that the differences between rate coefficients were mostly accounted for by differences between rats in tissue sensitivities to insulin. Thus in each 24h-starved rat, sleeping or resting, the main regulators of the plasma glucose concentrations were the rate of supply of gluconeogenic substrates from energy metabolism and the intrinsic sensitivity of the tissues to insulin. 8. We found that a commonly used deionization method of purifying glucose for determination of its specific radioactivity was inadequate.