Equivalence of cell survival data for radiation dose and thermal dose in ablative treatments: analysis applied to essential tremor thalamotomy by focused ultrasound and gamma knife.

Thermal dose and absorbed radiation dose have historically been difficult to compare because different biological mechanisms are at work. Thermal dose denatures proteins and the radiation dose causes DNA damage in order to achieve ablation. The purpose of this paper is to use the proportion of cell survival as a potential common unit by which to measure the biological effect of each procedure. Survival curves for both thermal and radiation doses have been extracted from previously published data for three different cell types. Fits of these curves were used to convert both thermal and radiation dose into the same quantified biological effect: fraction of surviving cells. They have also been used to generate and compare survival profiles from the only indication for which clinical data are available for both focused ultrasound (FUS) thermal ablation and radiation ablation: essential tremor thalamotomy. All cell types could be fitted with coefficients of determination greater than 0.992. As an illustration, survival profiles of clinical thalamotomies performed by radiosurgery and FUS are plotted on a same graph for the same metric: fraction of surviving cells. FUS and Gamma Knife have the potential to be used in combination to deliver a more effective treatment (for example, FUS may be used to debulk the main tumour mass, and radiation to treat the surrounding tumour bed). In this case, a model which compares thermal and radiation treatments is valuable in order to adjust the dose between the two.

Chronic treatment with D-chiro-inositol prevents autonomic and somatic neuropathy in STZ-induced diabetic mice.

AIM: D-chiro-inositol (DCI) has been shown to prevent and reverse endothelial dysfunction in diabetic rats and rabbits. The present study evaluates the preventive effect of DCI on experimental diabetic neuropathy (DN). METHODS: Streptozotocin-induced (STZ) diabetic mice were treated by oral gavage for 60 days with DCI (20 mg/kg/12 h) or saline (NaCl 0.9%; 0.1 ml/10 g/12 h; Diab) and compared with euglycaemic groups treated with saline (0.1 ml/10 g/12 h; Eugly). We compared the response of the isolated sciatic nerve, corpora cavernosa or vas deferens to electrical stimulation. RESULTS: The electrically evoked compound action potential of the sciatic nerve was greatly blunted by diabetes. The peak-to-peak amplitude (PPA) was decreased from 3.24 ± 0.7 to 0.9 ± 0.2 mV (p < 0.05), the conduction velocity (CV) of the first component was reduced from 46.78 ± 4.5 to 26.69 ± 3.8 ms (p < 0.05) and chronaxy was increased from 60.43 ± 1.9 to 69.67 ± 1.4 ms (p < 0.05). These parameters were improved in nerves from DCI-treated mice (p < 0.05). PPA in the DCI group was 5.79 ± 0.8 mV (vs. 0.9 ± 0.2 mV-Diab; p < 0.05) and CV was 45.91 ± 3.6 ms (vs. 26.69 ± 3.8 ms-Diab; p < 0.05). Maximal relaxation of the corpus cavernosum evoked by electrical stimulation (2-64 Hz) in the Diab group was 36.4 ± 3.8% compared to 65.4 ± 2.8% in Eugly and 59.3 ± 5.5% in the DCI group (p < 0.05). Maximal contraction obtained in the vas deferens was 38.0 ± 9.2% in Eugly and 11.5 ± 2.6% in Diab (decrease of 69.7%; p < 0.05), compared to 25.2 ± 2.3% in the DCI group (p < 0.05 vs. diabetic). Electron microscopy of the sciatic nerves showed prevention of neuronal damage. CONCLUSIONS: DCI has a neuroprotective action in both autonomic and somatic nerves in STZ-induced DN.

Phosphatidylinositol-glycan anchors of membrane proteins: potential precursors of insulin mediators.

BC3H1 myocytes release membrane-bound alkaline phosphatase to the incubation medium upon stimulation with insulin, following a time course that is consistent with the generation of dimyristoylglycerol and the appearance of a putative insulin mediator in the extracellular medium. The use of specific blocking agents shows, however, that alkaline phosphatase release and dimyristoylglycerol production are independent processes and that the blockade of either event inhibits the production of insulin mediator. These experiments suggest a new model of insulin action.

Generation by insulin of a chemical mediator that controls protein phosphorylation and dephosphorylation.

Deproteinized skeletal muscle extracts free of major nucleotides from control and insulin-treated rats were fractionated and assayed for inhibition of protein phosphorylation by cyclic adenosine monophosphate (AMP)-dependent and -independent protein kinases. A differential effect of insulin on a particular fraction was observed on cyclic AMP-dependent protein kinase but not on cyclic AMP-independent protein kinases. This fraction that inhibited cyclic AMP-dependent protein kinase also stimulated glycogen synthase phosphoprotein phosphatase. It is proposed that this fraction may contain a mediator substance generateed in the presence of insulin.

Radiation effects on DNA synthesis in a defined chromosomal replicon.

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G1/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.

Histone H1 and H3 dephosphorylation are differentially regulated by radiation-induced signal transduction pathways.

We recently demonstrated that linker histone H1, which is thought to have a fundamental role in higher-order chromatin structure, becomes transiently dephosphorylated after ionizing radiation (IR) in a mutated ataxia telangiectasia (ATM) dependent manner. To establish whether H1 dephosphorylation was a component of a damage-response pathway that included dephosphorylation of other histones, we asked whether H3 was dephosphorylated in response to IR in a manner similar to H1. H1 and H3 are maximally phosphorylated in metaphase and both are dephosphorylated after IR. However, the duration of IR-induced H3 dephosphorylation is significantly longer than that of IR-induced H1 dephosphorylation. Moreover, H1 dephosphorylation is ATM-dependent, whereas H3 dephosphorylation is ATM-independent. These observations suggest that the damage-sensing pathways regulating H3 and H1 dephosphorylation diverge upstream of ATM.

Epinephrine effects on cyclic AMP-dependent protein kinases from rat diaphragms.

Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.

Hormonal regulation of glycogen synthase: insulin decreases protein kinase sensitivity to cyclic AMP.

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1--2 microM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 microM. Insulin (0--100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.

Activation of liver guanylate cyclase by bile salts and contaminants in crude secretin and pancreozymin preparations.

Crude preparations of secretin or pancreozymin increased and at higher concentrations decreased guanylate cyclase (GTP pyophosphate-lyase, EC 4.6.1.2) activity from soluble and particulate fractions of rat liver homogenates. Partially purified and synthetic secretin were without effect as was the biologically active octapeptide fragment of pancreozymin. The active contaminants in these preparations survived boiling, saponification, and treatment with phospholipase A, trypsin and neuraminidase C. The activity was extractable with chloroform/methanol and did not survive ashing. Eight bile salt contaminants in crude secretin were obtained with thin-layer chromatography. Two of the contaminating bile salts that increased liver particulate guanylate cyclase activity were identified as taurodeoxycholate and either glycochenodeoxycholate or glycodeoxycholate; taurocholate was inhibitory. The sodium salts of cholate, deoxycholate, chenodeoxycholate and their glycine-or taurine-conjugated forms either increased or decreased particulate and soluble rat liver guanylate cyclase activity depending upon their concentration. Thus, the previously reported stimulatory and inhibitory effects of secretin and pancreozymin preparations on guanylate cyclase activity are probable attributable to their bile salt contaminants.

Actions of insulin-potentiating peptides on glycogen synthesis.

The action of urinary and synthetic AcG (acceleratory factor from growth hormone) peptides was studied in vitro and in vivo. Both peptides were inactive alone and active only in the presence of insulin to enhance glucose uptake, glycogen synthesis, and glycogen synthase conversion to the active I form in vitro and in vivo. Responses were dependent on both peptide and insulin concentrations in a dose-dependent manner. No response was obtained with glucose alone, but the presence of glucose did enhance the response of insulin alone or insulin in the presence of peptide. It is concluded that both AcG peptides enhance either the effective concentration or the activity of insulin at its site of action.

Studies on the insulin mediator. II. Separation of two antagonistic biologically active materials from fraction II.

Insulin treatment significantly altered the elution profile of deproteinized muscle extracts chromatographed on Sephadex G-25 columns, particularly in fraction II, which contains the insulin mediator. Further purification of fraction II by high-voltage paper electrophoresis at pH 1.9 and 3.5 resulted in two active fractions. Fraction 1 leads to 4 stimulated the cyclic AMP-dependent protein kinase and inhibited glycogen synthase phosphoprotein phosphatase, and may be a novel substance. Fractions 1 leads to 6 and 3 leads to 6 inhibited the cyclic AMP-dependent protein kinase and stimulated glycogen synthase phosphatase. It is proposed that the insulin mediator is present in fractions 1 leads to 6 and 3 leads to 6.

Insulin-like and insulin-enhancing effects of the sulfonylurea glyburide on rat adipose glycogen synthase.

The effects of long-term exposure of cultured rat adipose tissue to glyburide were examined on glycogen synthase activity. Glyburide alone caused an increase in the activity ratio (low glucose-6-P/high glucose-6-P) of glycogen synthase, and enhanced insulin's activation of the enzyme. The glyburide effects were time dependent, requiring fat pieces to be exposed to the drug for at least 10-20 h. The glucose concentration in the culture medium was also important: optimal concentrations of glucose were 10-20 mM. Glyburide acted to shift the insulin dose-response curve to the left by a factor of 2.5, but did not enhance the effects of maximal concentrations of the hormone. The Ka of the glyburide effects was about 2.0 microM. If glucose was omitted during the 20-min incubation with or without insulin, the increase in the activity ratio of glycogen synthase by glyburide was unaffected, but the enhancement of insulin action was reduced. Because these data indicate that glyburide's actions are glucose dependent, we propose that the sulfonylurea is probably acting to increase glucose transport, thus allosterically increasing the activity of a synthase phosphatase by glucose-6-P. The net result of this would be increased dephosphorylation and activation of glycogen synthase.

Insulin stimulation of glycogen synthase in cultured human diploid fibroblasts.

The effect of insulin on glycogen synthase activity in human diploid fibroblasts has been studied. As little as 2 X 10(-10) M insulin increased the glycogen synthase / activity without changing the total activity. Stimulation occurred within 5 min and became maximal in 30 min. A half-maximal increase of / activity was achieved at 3 X 10(-9) M insulin. Glucose starvation increased the magnitude of response of glycogen synthase to insulin but did not change the insulin concentration necessary to give a half-maximal stimulation. Glucose increased the basal level of / activity in human diploid fibroblasts; the effect of insulin was additive. During in vitro senescence the total glycogen synthase activity declined, but the concentration of insulin that produced a half-maximal stimulation remained unchanged. These data indicate that regulation of glycogen synthase activity in human diploid fibroblasts is responsive to physiologic insulin levels and that the system provides a useful model for the in vitro study of insulin sensitivity.

Effect of pH and 3-hydroxybutyrate on insulin binding and action in cultured human fibroblasts.

The influence of pH and 3-hydroxybutyrate on insulin binding and action has been studied in cultured human fibroblasts. When the pH of the incubation medium was decreased from 7.6 to 6.8, insulin stimulation of glycogen synthase and total glucose uptake was decreased. The decreased pH induced both an increase in the insulin concentration for half-maximal response, and a decrease in maximal responsiveness. When insulin binding was measured at lower pH, receptor affinity was decreased. The effect of 3-hydroxybutyrate on insulin binding and action was assessed at pH 7.4 and 6.9. In the presence of 3-hydroxybutyrate, insulin binding increased, but insulin action on glycogen synthase and total glucose uptake was unaffected. The data show that insulin action is impaired at lower pH. The decreased sensitivity is probably related to the decrease in insulin binding affinity at lower pH, but decreased maximal responsiveness implies that postreceptor components are also affected by lower pH. The results also suggest that 3-hydroxybutyrate induced a dissociation between binding and response, since an increase of insulin binding was not accompanied by changes in the regulation of glycogen synthase and total glucose uptake.

Mechanisms of aluminum fluoride- and insulin-stimulated p33 mRNA accumulation in rat hepatoma cells: involvement of a G protein and kinase action and demonstration of effects on mRNA turnover.

The insulin signalling pathway to control nuclear p33 gene expression was examined. An AlF4-stimulated pertussis toxin-insensitive G protein was shown to be involved. The action of AlF4- was completely blocked by deferoxamine. Insulin action was markedly stimulated in the presence of AlF4-. cAMP and diacylglycerol concentrations were examined as possible regulators but no increases were detected. The effects of AlF4- and of insulin were completely inhibited by the general kinase inhibitor H-7. A second calcium calmodulin protein kinase inhibitor, W-7, had no detectable effect. Insulin and AlF4- were shown to stabilize p33 mRNA.

Development of a cumulative irritation model for incontinence-associated dermatitis.

Incontinence-associated dermatitis (IAD) is a painful yet preventable form of cumulative skin irritation prevalent amongst those with limited movement. Consequently, it has a significant impact on the quality of life for those affected as well as substantial cost implications. Prevention and intervention is typically through good skin hygiene regimes and regular use of barrier products. In this paper, we describe the development of an in vivo model of IAD in healthy volunteers by occluded application of alkaline synthetic urine to the volar aspect of volunteer's forearms for 6 h per day over a five-day period to reproduce the moist and irritant conditions causative of IAD. Irritation was assessed and quantified on a daily basis by a series of non-invasive biophysical measurements and compared to a contralateral saline-treated (control) site. Dermal irritation was assessed by subjective (visual) and objective measurements (laser Doppler and polarisation spectroscopic imaging, infrared thermography, skin reflectance spectroscopy, transepidermal water loss and skin surface pH). The provocation of reproducible, cumulative skin irritation was successfully demonstrated and quantified. This five-day model of irritation is considered appropriate for the initial clinical assessment of topical products to prevent or treat IAD.

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes.

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).

The clearance and metabolism of estradiol and estradiol-17-esters in the rat.

The C-17 fatty acid esters of estradiol are naturally occurring estrogens which have been shown to circulate in blood. They are long-acting estrogens, analogous to the synthetic alkyl and aryl esters of estrogens which have been used pharmacologically for decades. To determine the mechanisms involved in the prolonged stimulation evoked by these nonpolar estrogens, several C-17 alkyl esters were synthesized and labeled with 3H at C-17 alpha, and their metabolism and clearance were studied and compared to those of estradiol in rats. The conversion of the C-17-3H to 3H2O was used as a marker of metabolism. While the clearance of the long chain esters from blood is somewhat slower than that of estradiol (t 1/2 = approximately 16 vs. 2 min, respectively), the rates of metabolism are dramatically different. For example, the t 1/2 of metabolism for two representative esters, estradiol-17-stearate and arachidonate, are 580 and 365 min, respectively, while the t 1/2 of metabolism for estradiol is about as fast as its clearance from blood (approximately 2 min). When the effect of chain length was studied, it was found that for the smaller esters, there was an inverse relationship between the size of the acyl group and the clearance from blood, i.e. the longer carboxylic acids were cleared more slowly. However, when the acyl group was lengthened from C12 to C14, the rate of clearance increased and was even faster with C18. Nevertheless, with all of the esters tested, the rate of metabolism steadily decreased as the chain length increased. These results are interpreted as indicating that the control point or rate-limiting step in the metabolism of the estradiol esters is the esterase that hydrolyzes the ester to estradiol. Thus, the prolonged estrogenic action of the C-17-alkyl esters is due to the slow release of estradiol from this hydrophobic reservoir.

Binding of estradiol-17-fatty acid esters to plasma proteins.

The C-17 fatty acid esters of estradiol are a unique family of long-acting estrogens that circulate in blood. The unusual duration of the estrogenic action of these esters has been shown previously to correlate with their very slow rate of metabolism. However, in striking contrast to their slow rate of metabolism, the clearance of these esters from blood is relatively rapid, not very different from that of estradiol (E2). Studies on the effect of the size of the carboxylic acid moiety on the rates of both metabolism and clearance have suggested that an active process might be involved in the cellular uptake of these circulating esters, and this, in turn, raised the question of how E2-fatty acid esters are transported in blood. The binding of representative E2-17-fatty acid esters to both human and rat plasma proteins known to bind either E2 or fatty acids was investigated. As expected, both E2 and 5 alpha-dihydrotestosterone bound to human sex hormone-binding globulin, whereas none of the E2 esters bound to this human plasma protein. Similarly, E2 bound to rat alpha-fetoprotein (AFP) and unsaturated fatty acids bound to both human and rat AFP, but none of the E2 esters bound to AFP of either species. These steroid esters bind to lipoproteins. Over 85% of a representative ester, E2-17-stearate, partitioned in the lipoprotein fractions of both human and rat serum, while the synthetic short chain ester, E2-17 beta-acetate, like E2 itself, partitioned predominantly in the nonlipoprotein fraction of blood. These results demonstrate the unusual binding of a family of steroid hormones to plasma lipoproteins and open the possibility that they are transported into target cells through the mediation of lipoprotein receptors.

Positive interaction between insulin and phorbol esters on the regulation of a specific messenger ribonucleic acid in rat hepatoma cells.

With Northern blot analysis, we demonstrate that phorbol 12-myristate 13-acetate (PMA) stimulated P-33 messenger RNA (mRNA)-accumulation in rat hepatoma cells in a time- and concentration-dependent manner similar to insulin and plant lectins. No effect of PMA on P-33 mRNA half-life was detected when mRNA synthesis was inhibited with either actinomycin D or 5.6-dichloro-1-beta-D-ribofuranosyl benzimidazole. The effects of insulin and PMA were additive at submaximal concentrations and no additivity was observed under these conditions at maximal concentrations. Thus PMA has a marked insulin-like effect on the accumulation of P-33 mRNA in rat hepatoma cells.