WAND2-A versatile wide angle neutron powder/single crystal diffractometer.

Wide Angle Neutron Diffractometer Squared is a high-flux versatile diffractometer with a 2-Dimensional Position Sensitive Detector at the High Flux Isotope Reactor. The instrument has strengths in both powder and single crystal diffraction. It is a unique instrument in the neutron scattering landscape of North America, and its capabilities are at least equal to similar instruments in the world.

Variant Philadelphia translocations in chronic myeloid leukaemia can mimic typical blast crisis chromosome abnormalities or classic t(9;22): a report of two cases.

A range of fluorescent in situ hybridization techniques have been used to reveal hidden variant Philadelphia translocations in two cases of Ph-positive chronic-phase chronic myeloid leukaemia. In one patient, a highly complex variant Ph translocation affecting four chromosomes had resulted in the formation of structures with the appearance of i(17q) and +8. Misinterpretation of these karyotypes has direct clinical relevance. Our findings illustrate that even established cytogenetic abnormalities may contain cryptic abnormalities beyond the resolution of conventional cytogenetic methods.

Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes.

Peptidic glucagon antagonists have been shown to lower blood glucose levels in diabetic models (1-3), but attempts to identify small molecular weight glucagon receptor-binding antagonists have met with little success. Skyrin, a fungal bisanthroquinone, exhibits functional glucagon antagonism by uncoupling the glucagon receptor from adenylate cyclase activation in rat liver membranes (1). We have examined the effects of skyrin on cells transfected with the human glucagon receptor and on isolated rat and human hepatocytes. The skyrin used was isolated from Talaromyces wortmanni American Type Culture Collection 10517. In rat hepatocytes, skyrin (30 micromol/l) inhibited glucagon-stimulated cAMP production (53%) and glucose output (IC50 56 micromol/l). There was no detectable effect on epinephrine or glucagon-like peptide 1 (GLP-1) stimulation of these parameters, which demonstrates skyrin's selective activity. Skyrin was also evaluated in primary cultures of human hepatocytes. Unlike cell lines, which are largely unresponsive to glucagon, primary human hepatocytes exhibited glucagon-dependent cAMP production for 14 days in culture (EC50 10 nmol/l). Skyrin (10 micromol/l) markedly reduced glucagon-stimulated cAMP production (55%) and glycogenolysis (27%) in human hepatocytes. The inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones. Skyrin is the first small molecular weight nonpeptidic agent demonstrated to interfere with the coupling of glucagon to adenylate cyclase independent of binding to the glucagon receptor. The data presented in this study indicate that functional uncoupling of the human glucagon receptor from cAMP production results in metabolic effects that could reduce hepatocyte glucose production and hence alleviate diabetic hyperglycemia.

Toward general methods of targeted library design: topomer shape similarity searching with diverse structures as queries.

A promising strategy for selecting synthetic targets is similarity-based searching of very large "virtual libraries", which comprise all structures accessible by linking two or three commercially available building blocks with combinatorial syntheses. To assess the general applicability of this strategy, leading structures taken from each of 34 recent medicinal chemistry publications were used as queries to search a virtual library containing 2.6 x 10(13) products from seven reactions, using a topomer shape similarity metric. Eighty-five percent of these searches succeeded, by yielding, with a search radius no greater than 120 topomer shape units, either at least 400 hits or hits from at least six sublibraries. From these 34 sets of search results, 122 representative structures were selected, illustrating potential "lead hops", or otherwise novel structures. Overall shape similarity to the query structure was confirmed for up to 95% of these representative structures, according to FLEXS, an algorithmically distinct program. Experimentally, there were 28 structures among those reported in the 34 query publications that were identified within the virtual library. Among these, the frequency of high activity was 87% for the 16 structures whose similarity to their query was 90 topomer units or less, compared to a frequency of 50% for the other 12 structures.

Cytogenetics of the chronic myeloid leukemia-derived cell line K562: karyotype clarification by multicolor fluorescence in situ hybridization, comparative genomic hybridization, and locus-specific fluorescence in situ hybridization.

The transformation of chronic myeloid leukemia (CML) from a chronic phase to an acute phase is frequently accompanied by additional chromosome changes. Extensive chromosome G-banded studies have revealed the secondary changes are nonrandom and frequently include trisomy 8, isochromosome 17q, trisomy 19, or an extra copy of the Philadelphia chromosome. In addition to these secondary chromosome changes, complex structural rearrangements often occur to form marker structures that remain unidentified by conventional G-banded analysis. The CML-derived cell line, K562, has been widely used in research since it was originally established in 1975. The K562 karyotype however, has remained incomplete, and marker structures have never been fully described. Recent advances in fluorescence in situ hybridization (FISH) technology have introduced the possibility of chromosome classification based on 24-color chromosome painting (M-FISH). In this study, we report a clarified karyotype for K562 obtained by a combination of the following molecular cytogenetic techniques: comparative genomic hybridization (CGH), FISH mapping using locus-specific probes, and M-FISH. Multicolor FISH has identified the marker structures in this cell line. The characteristic marker chromosome in K562 has been confirmed by this study to be a der(18)t(1;18). Multicolor FISH confirmed the identity of marker structures partially identified by G-banding as der(6)t(6;6),der(17)t(9;17),der(21)t(1;21),der(5)t(5;6). In addition M-FISH has revealed a deleted 20q and a complex small metacentric marker comprised of material from chromosomes 1, 6, and 20. A cryptic rearrangement was revealed between chromosomes 12 and 21 that produced a structure that looks like a normal chromosome 12 homologue by G-banding analysis. Finally, M-FISH detected regions from chromosome 13 intercalated into two acrocentric markers.

Molecular dissection of zyxin function reveals its involvement in cell motility.

Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.

Biochemical variants of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz (SLO or RSH) syndrome is characterized by multiple congenital anomalies, mental retardation, and defective growth; it results from an inherited defect in the biosynthesis of cholesterol. Patients have elevated plasma concentrations of 7-dehydrocholesterol, the immediate biosynthetic precursor of cholesterol and most also have low circulating levels of cholesterol. To understand better the biochemical basis of clinical variability, we evaluated cholesterol biosynthesis in lymphoblasts from 3 unrelated SLOS patients with distinct phenotypes. One patient has "type I SLOS", the second has the more severe "type II SLOS" and the third is classified as atypical and had been postulated to have a defect in sterol transport. The lymphoblasts of each patient show normal subcellular localization of cholesterol and 7-dehydrocholesterol by gradient fractionation. Biochemical differences in the ability of the lymphoblasts to convert 7-dehydrocholesterol to cholesterol are described and correspond to the severity of disease (type II > type I > atypical). Recently, the gene responsible for most SLOS cases (DHCR7) was mapped to chromosome 11 and mutations in DHCR7 were found in each of these patients. The biochemical differences described here likely result from the different mutations observed in DHCR7.

Possibility of distinct insulin-signaling pathways beyond phosphatidylinositol 3-kinase-mediating glucose transport and lipogenesis.

The aim of this study was to compare the effects of insulin and the insulinomimetic agent, englitazone, on functional end points and putative mediators of insulin action in 3T3-L1 adipocytes. Cells were incubated with englitazone for 48 h or with insulin for 10 or 30 min, or both, and 2-deoxy-D-[3H]glucose (2DG) uptake and lipogenesis (from [14C]glucose) were measured. Tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and pp60, and phosphatidylinositol (PI) 3-kinase activity (using PI as substrate) and mitogen-activated protein kinase (MAPK) activity were assayed in cell lysates. Englitazone increased 2DG uptake in a concentration-dependent (10-100 micromol/l) manner by up to sixfold, and preincubation with englitazone significantly enhanced insulin-stimulated 2DG uptake. However, englitazone had a biphasic effect on lipogenesis (163 +/- 13% basal at 10 micromol/l vs. 96 +/- 14% at 100 micromol/l), but when acetate was used as substrate, only concentration-dependent inhibition of lipogenesis occurred. In addition, englitazone decreased insulin-stimulated lipogenesis in a concentration-dependent manner. Englitazone did not increase IR, IRS-1/IRS-2, pp60, or MAPK phosphorylation, nor did it enhance insulin's stimulation of these parameters. Although englitazone alone did not activate PI 3-kinase, it did enhance the stimulation of the enzyme produced by a submaximally effective insulin concentration. Significant (63%) inhibition of insulin-stimulated lipogenesis occurred at a concentration of englitazone (30 micromol/l) that did not affect MAPK activation, which suggests that the drug's inhibitory effect on lipogenesis is not mediated by this pathway. Englitazone did not affect the expression of the peroxisome proliferator response element-containing fatty acyl CoA synthase gene, although it cannot be ruled out that expression of other lipogenic enzymes are altered by englitazone via peroxisome proliferator activated receptor-gamma activation or by an alternate pathway. Thus englitazone stimulates 2DG uptake without affecting PI 3-kinase, but it can enhance both insulin-stimulated 2DG uptake and PI 3-kinase activity. However, englitazone inhibits insulin-stimulated lipogenesis without inhibiting PI 3-kinase activity. Assuming activation of PI 3-kinase mediates insulin-stimulated 2-DG and lipogenesis, then the signaling pathways for each process diverge beyond PI 3-kinase.

Structure-function analysis of a series of glucagon-like peptide-1 analogs.

We have used NMR in conjunction with measurements of functional bioactivity to define the receptor-binding structure of glucagon-like peptide-1 (GLP-1.) Identification of the important residues for binding was accomplished by the substitution of amino acids at sites that seemed likely, from an examination of the amino acid sequence and from previously published observations, to be important in the three-dimensional (3D) structure of the molecule. Identification of the receptor-bound conformation of GLP-1, because it is a flexible peptide, required constraint of the peptide backbone into a predetermined 3D structure. Constraint was achieved by the introduction of disulfide bonds and specific side chain-side chain cross-links. The biological relevance of the synthetic structure of each rigidified peptide was assessed by measurement of its ability to bind to the receptor present on RINm5F cells and to elicit a functional response, cyclic AMP production. NMR solution structures were obtained for the most biologically relevant of these analogs. The results of this study indicated that the residues necessary for the biological activity of GLP-1 occupy approximately three equally-spaced regions of the peptide 3D structure, at the corners of an equilateral triangle whose sides are, at a minimum estimate, 12-15A.

Modulation of insulin secretion and glycemia by selective inhibition of cyclic AMP phosphodiesterase III.

The effects of selective inhibition of cyclic AMP phosphodiesterase type III on insulin and glucose levels during an oral glucose challenge were evaluated in obese, diabetic ob/ob mice and in lean, non-diabetic littermates using the selective inhibitor, milrinone. Oral administration of milrinone increased plasma insulin levels both in ob/ob and in lean mice. Glucose tolerance was improved in lean, but not in ob/ob mice, where glucose levels were increased by milrinone treatment. In isolated hepatocytes from normal rats incubation with 200 microM milrinone caused a 30% increase in glucose release with a corresponding depletion of glycogen stores. Stimulation of isolated rat adipocytes with 200 microM milrinone increased glycerol release 7-fold. We conclude that selective inhibitors of cyclic AMP phosphodiesterase III are effective insulin secretagogues, but their therapeutic utility may be limited by their concurrent stimulation of lipolysis and hepatic glucose output.

Comparison of the glucose dependency of glucagon-like peptide-1 (7-37) and glyburide in vitro and in vivo.

The purpose of the present study was to compare the glucose dependency of the insulin secretagogue activity of the sulfonylurea, glyburide, versus that of glucagon-like peptide-1(7-37) [GLP-1(7-37)] in vitro and in vivo. In freshly isolated rat islets, maximally effective concentrations of glyburide (10 micromol/L) and GLP-1(7-37) (10 nmol/L) were equally effective in stimulating insulin secretion in the presence of 15 mmol/L glucose (2.4-fold increase relative to 15 nmol/L glucose alone). At 5 nmol/L glucose, both agents increased insulin secretion, but the effect for glyburide was threefold greater than for GLP-1(7-37) (122% and 41% increase in insulin secretion, respectively). In conscious catheterized rats infused with glucose at a variable rate to clamp plasma glucose concentration at 11 mmol/L, glyburide (1 mg/kg orally) and GLP -1(7-37) (infused intravenously [IV] at 5 pmol/min/kg) produced similar increase in insulin levels (1.8-fold relative to the respective vehicle controls) that were sustained through 60 minutes of measurement. These doses of GLP-1(7-37) and glyburide were then administered to fasted and fed rats (basal plasma glucose concentration, 5.8 and 7.3 mmol/L, respectively). Relative to the vehicle control group, GLP-1(7-37) infusion produced a transitory increase (30%) in plasma insulin concentration and a modest sustained decrease (10% to 20%) in glucose in both fasted and fed rats, whereas glyburide induced a sustained 2.4- and 1.7-fold increase in plasma insulin concentration in fasted and fed rats, respectively, and a 50% decrease in plasma glucose in both fasted and fed rats. Results of these studies demonstrate the higher glucose threshold for the insulin secretagogue activity of GLP-1(7-37) relative to glyburide in vitro and in vivo.

The antihyperglycemic agent englitazone prevents the defect in glucose transport in rats fed a high-fat diet.

The effects of englitazone in male Wistar rats fed a high-fat diet (59% of calories as fat) were compared with control rats fed a high-carbohydrate diet (69% of calories as carbohydrate) (5-15 animals per group). Insulin-stimulated (17 nmol/l) 2-deoxy-D-glucose (2-DG) uptake was inhibited 31% in adipocytes isolated from rats on the high-fat diet for 3 weeks, but englitazone (50 mg/kg for the last 7 days) normalized the response. There was a selective decrease in GLUT4 (54 +/- 5% of high-carbohydrate) in epididymal fat from rats on the high-fat diet for 3 weeks, but englitazone treatment did not reverse the defect in GLUT4 (43 +/- 8% of high-carbohydrate) or increase GLUT1 (81 +/- 12% of high-carbohydrate). Englitazone normalized oral glucose (1 g/kg body wt) intolerance and excessive (210% of high-carbohydrate) liver glycogen deposition (from [14C]glucose) caused by the high-fat diet. The high-fat diet tended to decrease insulin receptor substrate-1 (IRS-1) and phosphatidylinositol-3'-kinase (PI-3-kinase) expression in epididymal fat (26% decrease; P < 0.1). Englitazone did not reverse this decrease in IRS-1 and PI-3-kinase levels in fat from high-fat-fed rats (there was a further 25-30% decrease, P < 0.05), nor did it increase PI-3-kinase activity in 3T3-L1 adipocytes under conditions (48 h incubation) where it stimulated 2-DG uptake sixfold or enhanced insulin-stimulated 2-DG uptake. In summary, englitazone prevented the insulin resistance associated with a high-fat diet, but the mechanism of action does not involve changes in fat or muscle glucose transporter content and may not involve activation of the insulin signaling pathway via PI-3-kinase.

Cyclic AMP phosphodiesterases of human and rat islets of Langerhans: contributions of types III and IV to the modulation of insulin secretion.

This study evaluated the contribution of isozymes of cAMP phosphodiesterase (PDE) to total PDE activity in human and rat islets using type-selective inhibitors. The effects of selected PDE inhibitors on insulin secretion from human and rat islets were also measured in order to assess the contribution of the various PDE isozymes to the modulation of insulin secretion. The data suggest that PDE III is quantitatively the most important PDE isozyme present in islets, accounting for up to 70% of the total activity. Lower, but measurable, levels of PDE IV activity were present. Approximately 20% of islet PDE is not inhibitable by agents selective either for PDE III or IV. Selective inhibition of PDE III stimulated insulin secretion, but inhibition of PDE IV had no effect. The effects of type-selective inhibitors on PDE activity and insulin secretion were similar in human and rat islets.

Chronic intrahypothalamic insulin infusion in the rat: behavioral specificity.

This study examines whether chronic intrahypothalamic (IH) insulin infusions suppress body weight and food intake directly or via effects on water intake or activity. Insulin (15 microU/h) was infused into the ventromedial hypothalamic nucleus of rats for 1 week. If IH insulin infusions primarily suppress water intake, animals should consume less water during insulin infusion in the absence of food. In the first experiment in this study, rats food deprived during IH insulin infusion did not drink significantly less than during vehicle infusion. This implies that IH insulin affects water intake secondarily to its impact on food intake. Insulin might suppress food intake and body weight by decreasing overall activity levels, including activity involved in ingestive behavior. In the second experiment, rats' activity on a running wheel was measured during IH insulin and vehicle infusion; activity increased during insulin infusion compared to vehicle infusion. These findings suggest that insulin's effects on food intake and body weight are via a mechanism that does not appear to directly influence water intake, and does not reduce overall activity levels.

Water intake during chronic preoptic infusions of osmotically active or inert solutions.

To further elucidate the role of the lateral preoptic area (LPO) as an osmoreceptive region, rats received chronic infusions (2 weeks) of low volumes (0.5 microliters/h) solutions of hypertonic sodium chloride (NaCl; 0.16 M), hypertonic potassium chloride (KCl; 0.16 M), hypertonic (0.32 M) or hypotonic (0.16 M) mannitol, isotonic saline, or water delivered bilaterally via subcutaneous osmotic minipumps attached to intracranial cannulae. All cannulae terminated within the anterior hypothalamus-preoptic region. Hypertonic NaCl and KCl increased water intake over preinfusion levels in the majority of animals tested. However, the effects were variable, including some sizable increases as well as decreases. Hypertonic mannitol decreased daily water intake in 15 of 25 rats and produced essentially no change in the average intake of the group. Isotonic NaCl produced smaller increases and decreases, while water produced larger changes in individual rats, but neither solution had a significant effect on the average intake of the group. None of the infusates significantly altered food intake.

Role of intrahypothalamic insulin in circadian patterns of food intake, activity, and body temperature.

These experiments examined the extent to which chronic intrahypothalamic (IH) insulin infusions that alter circadian patterns of food intake (FI) affect the regulation of other diurnally varying behavior in the rat. One-week IH insulin infusion (1.5 microU/hr) significantly decreases rats' night FI and increases day FI but does not alter the diurnal pattern of activity. Mean daily core temperature increased slightly but significantly during insulin infusion, the daily peak of the body temperature rhythm did not shift significantly, and the daily range of body temperature increased. IH insulin infusion in rats living in constant light and thus without circadian rhythm of FI led to significant decreases in FI and body weight. These data support the conclusion that IH insulin infusion alters food intake and body weight through a specific effect on a neural system that regulates food intake and body weight, and not by altering circadian rhythms.

Chronic intrahypothalamic infusions of insulin or insulin antibodies alter body weight and food intake in the rat.

In Experiment 1, one-week infusion of insulin (0.15, 1.5, or 15.0 microU/hr) into the ventromedial hypothalamus (VMH) of rats reduced body weight (BW) and nighttime food intake (FI). While 0.15 microU/h decreased daytime FI, 1.5 microU/h increased daytime FI and 15.0 microU/h left daytime FI unchanged. Total daily FI was decreased by the two highest doses. In Experiment 2, intra-VMH infusion of specific insulin antibodies (1.5 microUeq/h) increased BW and FI, while C-peptide antibodies were ineffective. In Experiment 3a, intracerebroventricular infusions of insulin failed to decrease FI and BW comparably to similar intrahypothalamic infusions. In Experiment 3b, intra-VMH insulin was infused via cannulae that bypassed the cerebral ventricles. The decrease in FI and BW was comparable to that observed when insulin was infused via cannulae that penetrated a ventricle. Histology from animals used in Experiments 1-3 indicates that optimum sites for insulin-induced changes in BW and FI in the hypothalamus lie in an area that includes portions of the paraventricular, arcuate, dorsomedial, and ventromedial nuclei.

Insulinlike activity of new antidiabetic agent CP 68722 in 3T3-L1 adipocytes.

We examined the in vitro effects of CP 68722, a novel antidiabetic agent, in 3T3-L1 adipocytes. CP 68722 stimulated 2-deoxyglucose uptake in the absence of insulin. At least 30 min of incubation were required for stimulation of uptake. This effect increased over 5 h and was sustained up to 72 h. The stimulation of 2-deoxyglucose uptake by CP 68722 could be inhibited approximately 60% by inhibition of protein synthesis with cycloheximide. Half-maximal and maximal responses to CP 68722 at 72 h of incubation were observed at 10 and 100 microM of drug, respectively, with a threefold stimulation of uptake at 100 microM approximating the maximal response of these cells to acute insulin stimulation. CP 68722 was able to overcome insulin resistance induced by dexamethasone in 3T3-L1 cells. The effect of drug, like that of insulin, was primarily to increase the Vmax of 2-deoxyglucose uptake. The stimulation of uptake by CP 68722 or insulin could be prevented by incubating the cells at 10 degrees C, a temperature that impedes translocation of glucose transporters to the plasma membrane. Therefore, it appears that CP 68722, like insulin, stimulates glucose uptake by a mechanism that involves translocation of intracellular glucose transporters to the plasma membrane and de novo protein synthesis. We compared the effect of CP 68722 with the sulfonylureas, the primary drugs used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). CP 68722 was a more potent and effective stimulator of 2-deoxyglucose uptake in 3T3-L1 cells than either first- or second-generation sulfonylureas.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic intrahypothalamic infusion of insulin on food intake and diurnal meal patterning in the rat.

In Experiment 1, rats were chronically infused with insulin (2.7, 27, or 270 ng/hr) or 0.9% saline into the ventromedial (VMH), medial perifornical (PF), or lateral (LH) hypothalamus. VMH infusions of insulin caused a significant, dose-dependent decrease in food intake and body weight; PF infusion of insulin was less effective, but significant; whereas LH infusions of insulin were ineffective. In Experiment 2, rats were chronically infused with insulin (0.54 ng/hr) or 0.9% saline into the VMH, paraventricular (PVN), or posterior (PN) hypothalamic nucleus. Subjects that received VMH or PN infusions of insulin failed to regain weight lost as a result of surgery even 2 weeks after infusion; subjects that received PVN infusions of insulin regained their preoperative weights faster than did controls. All of the groups that received insulin significantly increased their daytime food intake during the infusion period and decreased their night food intake slightly; 24-hr food intake remained unchanged.