Detection of Early Ischemic Changes with Virtual Noncontrast Dual-Energy CT in Acute Ischemic Stroke: A Noninferiority Analysis.

BACKGROUND AND PURPOSE: Dual-energy virtual NCCT has the potential to replace conventional NCCT to detect early ischemic changes in acute ischemic stroke. In this study, we evaluated whether virtual NCCT is noninferior compared with standard linearly blended NCCT, a surrogate of conventional NCCT, regarding the detection of early ischemic changes with ASPECTS. MATERIALS AND METHODS: Adult patients who presented with suspected acute ischemic stroke and who underwent dual-energy NCCT and CTA and brain MR imaging within 48 hours were included. Standard linearly blended images were reconstructed to match a conventional NCCT. Virtual NCCT images were reconstructed from CTA. ASPECTS was evaluated on conventional NCCT, virtual NCCT, and DWI, which served as the reference standard. Agreement between CT assessments and the reference standard was evaluated with the Lin concordance correlation coefficient. Noninferiority was assessed with bootstrapped estimates of the differences in ASPECTS between conventional and virtual NCCT with 95% CIs. RESULTS: Of the 193 included patients, 100 patients (52%) had ischemia on DWI. Compared with the reference standard, the ASPECTS concordance correlation coefficient for conventional and virtual NCCT was 0.23 (95% CI, 0.15-0.32) and 0.44 (95% CI, 0.33-0.53), respectively. The difference in the concordance correlation coefficient between virtual and conventional NCCT was 0.20 (95% CI, 0.01-0.39) and did not cross the prespecified noninferiority margin of -0.10. CONCLUSIONS: Dual-energy virtual NCCT is noninferior compared with conventional NCCT for the detection of early ischemic changes with ASPECTS.

Prediction of Clinical Outcome in Patients with Large-Vessel Acute Ischemic Stroke: Performance of Machine Learning versus SPAN-100.

BACKGROUND AND PURPOSE: Traditional statistical models and pretreatment scoring systems have been used to predict the outcome for acute ischemic stroke patients (AIS). Our aim was to select the most relevant features in terms of outcome prediction on the basis of machine learning algorithms for patients with acute ischemic stroke and to compare the performance between multiple models and the Stroke Prognostication Using Age and National Institutes of Health Stroke Scale (SPAN-100) index model. MATERIALS AND METHODS: A retrospective multicenter cohort of 1431 patients with acute ischemic stroke was subdivided into recanalized and nonrecanalized patients. Extreme Gradient Boosting machine learning models were built to predict the mRS score at 90 days using clinical, imaging, combined, and best-performing features. Feature selection was performed using the relative weight and frequency of occurrence in the models. The model with the best performance was compared with the SPAN-100 index model using area under the receiver operating curve analysis. RESULTS: In 3 groups of patients, the baseline NIHSS was the most significant predictor of outcome among all the parameters, with relative weights of 0.36∼0.69; ischemic core volume on CTP ranked as the most important imaging biomarker with relative weights of 0.29∼0.47. The model with the best-performing features had a better performance than the other machine learning models. The area under the curve of the model with the best-performing features was higher than SPAN-100 model and reached statistical significance for the total (P < .05) and the nonrecanalized patients (P < .001). CONCLUSIONS: Machine learning-based feature selection can identify parameters with higher performance in outcome prediction. Machine learning models with the best-performing features, especially advanced CTP data, had superior performance of the recovery outcome prediction for patients with stroke at admission in comparison with SPAN-100.

Insulin action in cultured human skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and GLUT1 content.

In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, cultured myotubes are a useful tool to facilitate biological and molecular validation of novel pharmacological agents aimed to improve glucose metabolism in skeletal muscle.

Discovery of a potent, highly selective, and orally efficacious small-molecule activator of the insulin receptor.

A series of 3,6-diaryl-2,5-dihydroxybenzoquinones were synthesized and evaluated for their abilities to selectively activate human insulin receptor tyrosine kinase (IRTK). 2, 5-Dihydroxy-6-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h) was identified as a potent, highly selective, and orally active small-molecule insulin receptor activator. It activated IRTK with an EC(50) of 300 nM and did not induce the activation of closely related receptors (IGFIR, EGFR, and PDGFR) at concentrations up to 30 000 nM. Oral administration of the compound to hyperglycemic db/db mice (0.1-10 mg/kg/day) elicited substantial to nearly complete correction of hyperglycemia in a dose-dependent manner. In ob/ob mice, the compound (10 mg/kg) caused significant reduction in hyperinsulinemia. A structurally related compound 2c, inactive in IRTK assay, failed to affect blood glucose level in db/db mice at equivalent exposure levels. Results from additional studies with compound 2h, aimed at evaluating classical quinone-related phenomena, provided sufficient grounds for optimism to allow more extensive toxicologic evaluation.

Activation of insulin signal transduction pathway and anti-diabetic activity of small molecule insulin receptor activators.

We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T. , Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974-977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.

Differential regulation of glycogen synthase kinase 3beta by insulin and Wnt signaling.

Glycogen synthase kinase 3beta (GSK3beta) is a key component in many biological processes including insulin and Wnt signaling. Since the activation of each signaling pathway results in a decrease in GSK3beta activity, we examined the specificity of their downstream effects in the same cell type. Insulin induces an increased activity of glycogen synthase but has no influence on the protein level of beta-catenin. In contrast, Wnt increases the cytosolic pool of beta-catenin but not glycogen synthase activity. We found that, unlike insulin, neither the phosphorylation status of the serine9 residue of GSK3beta nor the activity of protein kinase B is regulated by Wnt. Although the decrease in GSK3beta activity is required, GSK3beta may not be the limiting component for Wnt signaling in the cells that we examined. Our results suggest that the axin-conductin complexed GSK3beta may be dedicated to Wnt rather than insulin signaling. Insulin and Wnt pathways regulate GSK3beta through different mechanisms, and therefore lead to distinct downstream events.

Estradiol causes a dose-dependent stimulation of prostate growth in castrated beagle dogs.

BACKGROUND: Previous studies have shown that chronic treatment of castrate dogs with androgen and estrogen results in significant prostate growth. Estrogen treatment of castrate dogs in the absence of androgen has resulted in conflicting data as reported by several authors. The purpose of this experiment was to evaluate the effect of a physiological dose of estradiol on prostate growth in dogs, using ultrasound to study size changes over time. METHODS: Dogs (n = 25) were randomly divided into groups (n = 5) and treated as follows: castration alone (CC), castration plus low dose estradiol (E(2) low), castration plus high estradiol (E(2) high), castration plus estradiol and androstanediol (E(2)A), or no treatment (normal controls, NC). Silastic implants containing 5alpha-androstan-3alpha-17beta-diol (3alphadiol), and/or 17beta-estradiol were used for continous delivery of steroids. Prostate volume was measured by transrectal ultrasonography, and blood was drawn for hormone and sex hormone binding globulin (SHBG) determinations. RESULTS: Results show that serum estradiol and SHBG levels were fairly constant over 12 weeks in all groups. Estradiol-treated groups had mean serum estradiol values of approximately 40 and 60 pg/ml, respectively. Initially, all groups had similar prostate volumes. Over 12 weeks the castrate dogs had a decline in prostate volume, whereas the intact dogs and those treated with E(2) and 3alpha-diol maintained a constant prostate volume. Estradiol treatment caused a large, late onset (week 7), dose-dependent increase in prostate volume relative to the intact group (P < 0.01). At 12 weeks, animals were euthanized and prostates weighed. The mean prostate weights in each group were: NC 14.8 +/- 2. 9, CC 2.4 +/- 0.5, E(2)A 9.7 +/- 2.0, E(2) low 21.7 +/- 4.3, and E(2) high 63.6 +/- 12.6 g (geometric mean +/- SEM). Histologically, prostates of estrogen-treated dogs showed metaplastic squamous epithelium. CONCLUSIONS: These results demonstrate that estradiol causes marked dose-dependent stimulation of prostate growth in the castrate dog.

Sex hormone-binding globulin mediates prostate androgen receptor action via a novel signaling pathway.

Estradiol (E2) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) have been implicated in prostate hyperplasia in man and dogs, but neither of these steroids bind to androgen receptors (ARs). Recently, we reported that E2 and 3alpha-diol stimulated generation of intracellular cAMP via binding to a complex of sex hormone-binding globulin (SHBG) and its receptor (R(SHBG)) on prostate cells. We speculated that this pathway, involving steroids normally found in the prostate, was involved in the indirect activation of ARs. Using the dog as a model to test this hypothesis in normal prostate, we investigated whether E2, 3alpha-diol, and SHBG stimulated the production of the androgen-responsive protein, arginine esterase (AE), the canine equivalent of human prostate-specific antigen. In cultured dog prostate tissue preincubated with SHBG, E2 and 3alpha-diol stimulated AE activity. These effects were blocked by hydroxyflutamide, an AR antagonist, and by 2-methoxyestradiol, a competitive inhibitor of E2 and 3alpha-diol binding to SHBG. In the absence of exogenous steroids and SHBG, AE also was significantly increased by treatment with forskolin or 8-Bromoadenosine-cAMP. These observations support the hypothesis that in normal prostate, E2 and 3alpha-diol can amplify or substitute for androgens, with regard to activation of the AR via the R(SHBG) by a signal transduction pathway involving cAMP. Because both E2 and 3alpha-diol are involved in the pathogenesis of benign prostatic hyperplasia in dogs and implicated in benign prostatic hyperplasia in man, antagonism of the prostatic SHBG pathway may offer a novel and attractive therapeutic target.

5 alpha-Androstan-3 alpha,17 beta-diol is a hormone: stimulation of cAMP accumulation in human and dog prostate.

5 alpha-Androstan-3 alpha, 17 beta-diol (3 alpha-diol) is a well known metabolite of dihydrostestosterone (DHT) and the concentration of its glucuronide in serum is looked upon as a marker of DHT activity in skin and prostate. The data in this communication show that 3 alpha-diol is a potent agonist of the human and canine prostatic SHBG-[SHBG receptor]. It causes a robust rise in intracellular cAMP within minutes; the 1/2 max. response occurs at a concentration equal to its concentration in male plasma. Estradiol is the only other agonist in this system. All other steroids that bind to SHBG are antagonists.

Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli.

We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.

Regulation of microsomal, xenobiotic epoxide hydrolase messenger RNA in persistent hepatocyte nodules and hepatomas induced by chemical carcinogens.

We have utilized a DNA clone complementary to epoxide hydrolase mRNA as a probe to examine the level of the mRNA in persistent hepatocyte nodules and hepatomas induced by the Solt-Farber chemical carcinogenesis procedure. Epoxide hydrolase mRNA is increased 14-fold in nodules as compared to the level in normal liver. When rats with liver nodules were administered phenobarbital, an inducer of epoxide hydrolase mRNA in normal animals, a superinduction in epoxide hydrolase mRNA was observed in the nodules (22-fold) as compared to normal liver. Surprisingly, nodule induction in conjunction with phenobarbital administration also produced marked elevation in epoxide hydrolase mRNA levels in the tissue surrounding the nodules. Using HpaII and MspI to assess the degree of methylation of CCGG sites, we have found that the epoxide hydrolase gene is hypomethylated in nodules and hepatomas compared to the gene in normal liver tissue. Phenobarbital treatment alone increased epoxide hydrolase mRNA levels but did not result in hypomethylation of the epoxide hydrolase gene. These data further support the observation that hypomethylation of specific gene sequences occurs during chemical carcinogenesis and is correlated with an elevation in the steady state level of epoxide hydrolase mRNA in persistent hepatocyte nodules.

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells.

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.

Sequence analysis and regulation of rat liver glutathione S-transferase mRNAs.

We have utilized polysomal immunoadsorption techniques to purify the rat liver glutathione S-transferase mRNAs. Using the purified mRNAs as template, cDNA clones complementary to the Ya, Yb1, and Yc mRNAs have been constructed. The cDNA clones have been utilized in RNA blot hybridization and nuclear run-off assays to demonstrate that the Ya and Yb mRNAs are elevated 8 and 5-fold, respectively by phenobarbital; whereas the Yc mRNA is elevated only 2.0-fold. The elevation in glutathione S-transferase mRNAs is due in part to transcriptional activation of the corresponding genes. Nucleotide sequence analysis of the three glutathione S-transferase clones suggest that the Ya and Yc genes represent one rat liver glutathione S-transferase gene family whereas the Yb genes represent a second distinct glutathione S-transferase gene family. The construction of these cDNA clones will allow identification and characterization of the glutathione S-transferase structural genes as well as aid in the identification of regulatory elements that are responsible for transcriptional activation of the genes by xenobiotics.

Rat liver glutathione S-transferases. DNA sequence analysis of a Yb2 cDNA clone and regulation of the Yb1 and Yb2 mRNAs by phenobarbital.

We have constructed a cDNA clone, pGTA/C48, which is complementary to the rat liver glutathione S-transferase Yb2 mRNA. Recombinant clone pGTA/C48 contains a cDNA insert of 845 base pairs which overlaps nucleotides 108-952 of the Yb1 cDNA clone, pGTA/C44, described previously by our laboratory (Ding, G. J.-F., Lu, A. Y. H., and Pickett, C. B. (1985) J. Biol. Chem. 260, 13268-13271). Over the protein coding region of the Yb1 and Yb2 cDNA clones there is an 84% nucleotide sequence homology, whereas the 3' untranslated regions are only 32% homologous. The complete amino acid sequence of the Yb2 subunit has been determined from a combination of DNA sequence analysis of pGTA/C48 and conventional protein sequence analysis of the glutathione S-transferase Yb1 Yb2 heterodimer. The Yb2 subunit is comprised of 218 amino acids with a molecular weight of 25,705 and has an amino acid sequence which is 79% homologous to the sequence of the Yb1 subunit. We have utilized the divergent 3' untranslated regions of three rat liver glutathione S-transferase cDNA clones as specific probes to determine the effect of phenobarbital on the level of Yb1, Yb2, and Yc mRNAs. Our results clearly show that the Yb1 and Yb2 mRNAs are elevated approximately 5-6-fold by phenobarbital administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic. Finally, our data suggest that the Yb2 subunit is encoded by a gene(s) which is distinct from the Yb1 gene(s) and provides direct evidence for the existence of multiple glutathione S-transferase Yb genes in the rat.

Transcriptional regulation of rat liver glutathione S-transferase genes by phenobarbital and 3-methylcholanthrene.

The relative rates of transcription of the rat liver glutathione S-transferase Ya-Yc and Yb genes were determined in purified liver nuclei isolated at different times after phenobarbital or 3-methylcholanthrene administration. The transcriptional rates of the Ya-Yc and Yb genes were elevated approximately fivefold 8 and 6 h, respectively, after phenobarbital administration. In contrast, the transcriptional rates of the Ya-Yc genes were elevated approximately eightfold at 16 h after 3-methylcholanthrene administration, whereas the transcriptional rates of the Yb genes were elevated approximately fivefold at 6 h after the administration of this xenobiotic. The elevation in transcriptional activity of the glutathione S-transferase genes is sufficient to account for the increase in glutathione S-transferase mRNA levels determined previously by RNA blot hybridization [C. B. Pickett, C. A. Telakowski-Hopkins, G. J-F. Ding, L. Argenbright, and A. Y. H. Lu (1984) J. Biol. Chem. 259, 5182-5188]. Therefore, it appears that phenobarbital and 3-methylcholanthrene elevate the level of the rat liver glutathione S-transferases primarily by augmenting the transcriptional rates of their respective genes.